Peptides for treating and preventing immune-related disorders, including treating and preventing infection by modulating innate immunity

ABSTRACT

In one aspect, the present invention provides isolated novel peptides that can be used to modulate innate immunity in a subject and/or for the treatment of an immune-related disorder, including treating and preventing infection by modulating innate immunity. Also provided are an agent reactive with the peptide, a pharmaceutical composition that includes the peptide, an isolated nucleic acid molecule encoding the peptide, a recombinant nucleic acid construct that includes the nucleic acid molecule, at least one host cell comprising the recombinant nucleic acid construct, and a method of producing the peptide using the host cell. The present invention further provides a method for treating and/or preventing infection in a subject by administering the peptide of the invention to the subject, thereby modulating innate immunity in the subject. Additionally, the present invention provides a method for predicting whether a subject would be responsive to treatment with a peptide of the invention.

PRIOR RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/404,965, filed on Mar. 16, 2009 now abandoned, which is acontinuation of U.S. patent application Ser. No. 11/730,695, filed onApr. 3, 2007, now abandoned, which is a

national stage under 35 USC 371 of PCT/CA2006/001650, filed Oct. 4,2006, entitled, “Novel Peptides For Treating And PreventingImmune-Related Disorders, Including Treating And Preventing Infection ByModulating Innate Immunity”, which claims the benefit of the priority ofU.S. Provisional Patent Application No. 60/722,962, filed Oct. 4, 2005,U.S. Provisional Patent Application No. 60/722,958, filed Oct. 4, 2005and U.S. Provisional Patent Application No. 60/722,959, filed Oct. 4,2005. Each of these applications is incorporated by reference herein inits entirety.

FIELD OF THE INVENTION

This invention relates to peptides for use in treating and preventingimmune-related disorders, including treating and preventing infection bymodulating innate immunity. In one aspect, the invention relates tocompositions and uses thereof for modulating innate immunity. In anotheraspect, the invention provides novel peptides and uses thereof effectivein reducing dipeptidyle peptidase (DPPIV) activity.

BACKGROUND OF THE INVENTION

A variety of microorganisms, including viruses, bacteria, fungi, andparasites, can cause disease. Microbial cells are distinct from thecells of animals and plants which are unable to live alone in nature,existing only as parts of multicellular organisms. Microbial cells canbe pathogenic or non-pathogenic, depending, in part, on themicroorganism and the status of the host. For example, in animmunocompromised host, a normally harmless bacterium can become apathogen.

Drug resistance remains an obstacle in the ongoing effort to fightinfection. For example, penicillin was effective in treatingStaphylococcus aureus, until the bacterium became resistant. Throughoutthe second half of the 20^(th) century, new antibiotics, such asvancomycin and methicillin, were developed; these successfully cured S.aureus infections. However, methicillin-resistant strains of S. aureusevolved in the 1970s, and have been plaguing hospitals worldwide eversince. More recently, vancomycin-resistant strains of S. aureus havesurfaced.

With the increasing threat of resistance to antimicrobial drugs and theemergence of new infectious diseases, there exists a continuing need fornovel therapeutic compounds. Therapeutics that act on the host, not thepathogen, are desirable, because they do not encourage pathogenicresistance. In particular, drugs that act on the host via the innateimmune system provide a promising source of therapeutics.

Host defense against microorganisms begins with the epithelial bathersof the body and the innate immune system, and culminates in theinduction of the adaptive immune response. The host innate immuneresponse encompasses a set of highly-conserved mechanisms that recognizeand counter microbial infections. Elements of innate immunity arecontinuously maintained at low levels, and are activated very rapidlywhen stimulated. The innate immune response begins with events thatoccur immediately after exposure to a microbial pathogen. Eventsassociated with adaptive immunity, such as rearrangement ofimmunoglobulin receptor genes, are not considered part of the innateresponse.

There is evidence to indicate that innate responses are instrumental incontrolling most infections, and can also contribute to inflammatoryresponses. Inflammatory responses triggered by infection are known to becentral components of disease pathogenesis. The importance of Toll-likereceptors (TLRs) in the innate immune response has also been wellcharacterized. The mammalian family of TLRs recognizes conservedmolecules, many of which are found on the surfaces of, or are releasedby, microbial pathogens. There are numerous other mechanisms, less wellcharacterized, that initiate and/or contribute to the host innatedefense.

The innate immune system provides a range of protective mechanisms,including epithelial-barrier function and secretion of cytokines andchemokines. To date, four families of chemokines have been categorized,according to the number of conserved N-terminal cysteine motifs: C, CC,CXC, and CX3C, where X is a non-conserved amino acid residue. The CXCchemokines are known to be chemotactic for cells bearing the CXCR3receptor, including monocytes, activated T cells (Th1), and NK cells.Primary human airway epithelial cells, and the cell line 16-HBE,constitutively express the CXCR3 receptor and its ligands, JP-10, I-TAC,and MIG (Kelsen et al., The chemokine receptor CXCR3 and its splicevariant are expressed in human airway epithelial cells, Am. J. Physiol.Lung Cell Mol. Physiol., 287:L584, 2004). Furthermore, CXCR3 ligandsinduce chemotactic responses and actin reorganization in 16-HBE cells(Kelsen et al., The chemokine receptor CXCR3 and its splice variant areexpressed in human airway epithelial cells, Am. J. Physiol. Lung CellMol. Physiol., 287:L584, 2004).

Further, the type II transmembrane serine protease dipeptidyl peptidase(DPPIV), also known as CD26 or adenosine deaminase binding protein, is amajor regulator of various physiological processes including immunefunctions. CD26/DPPIV is a 110-kD cell surface glycoprotein that ismainly expressed on mature thymocytes, activated T-cells, B-cells,NK-cells, macrophages, and epithelial cells. It has at least twofunctions, a signal transduction function and a proteolytic function(Morimoto C, Schlossman S F. The structure and function of CD26 in theT-cell immune response. Immunol Review. 1998, 161: 55-70.). One of itscellular roles involves modulation of chemokine activity by cleavingdipeptides from the chemokine N-terminus. The modulation of the NH₂termini of chemokines is of great importance not only for binding totheir receptors and the following reactions but also for altering thereceptor specificity of the processed chemokine. DPPIV activity has beenassociated with a number of immune-related conditions.

SUMMARY OF THE INVENTION

The inventors have discovered that peptides having the amino acidsequence of one of the peptides listed and described in TABLE 1 (i.e.,SEQ ID NOS:1-90) or an analogue, derivative, variant or obvious chemicalequivalent thereof can enhance a host's innate immunity. In one aspect,the immunomodulatory peptides of the invention were found to lack directantimicrobial activity while demonstrating an ability to improvesurvival in infected hosts. In another aspect, the invention providespeptides that modulate DPPIV activity. In one aspect the inventionprovides peptides that reduce DPPIV activity. In yet another aspect, theinvention provides peptides which can be used in the diagnosis,treatment or prevention of an immunological disorder, such as oneassociated with DPPIV activity and/or innate immunity.

Accordingly, in one aspect, the present invention provides an isolatedpeptide that includes the amino acid sequence of any one of SEQ IDNOS:1-90 or an analogue, derivative, or variant thereof or obviouschemical equivalent thereof or a peptide comprising said peptide. In oneembodiment the peptide is up to 7, 8 9, or 10 amino acids comprisingsaid peptide of SEQ ID NOS:1-43, 45-53, and 55-90 or analogue,derivative, variant or obvious chemical equivalent thereof. In oneembodiment, the peptide is up to 7 amino acids comprising SEQ IDNOS:1-43, 45-53, and 55-90. In another embodiment, it is a peptidecomprising a peptide of SEQ. ID. Nos 1, 3-16, 18-43, 45-53 or 55-90 oranalogue, derivative, variant or obvious chemical equivalents thereof orpeptide of up to 7, 8, 9, or 10 amino acids comprising same. By way ofexample, the isolated peptide may have a modified C-terminus (e.g., anamidated C-terminus) and/or a modified N-terminus. The isolated peptideof the invention may further include an amino acid sequence of TABLE 1(SEQ ID NOS:1-90) or analogue, derivative, variant or obvious chemicalequivalent thereof as modified by at least one substitution of a D aminoacid. The isolated peptide may further include a modified backbone, byway of example, wherein the N-terminus is modified from an amide to anN-methyl. In one aspect, those modified peptides which retain theimmunological activity of the parent peptide and obvious chemicalequivalent thereto which retain said activity are encompassed within thescope of the present invention.

In another aspect, the present invention further provides an agentreactive with an isolated peptide that includes the amino acid sequenceof TABLE 1 or an analogue, derivative, or variant thereof. In oneembodiment, the agent is a non-naturally occurring antibody (e.g., apolyclonal or monoclonal antibody). In one embodiment, the antibody ismade using a MAPS Antigen (Tam P J (1988). Synthetic peptide vaccinedesign: Synthesis and properties of a high-density multiple antigenicpeptide system. Proc Natl Acad Sci 85, pp. 5409-5413., Briand J P, DarinC, Van Regenmortel M H W, Muller S (1992). Application and limitationsof the multiple antigen peptide (MAP) system in the production andevaluation of anti-peptide and anti-protein antibodies. J Immunol Meth156:2, pp. 255-265) attached to the peptide of the present invention via2 glycine residues inserted at the C-terminus of the peptide. Theconstruct can then be administered to an animal, such as a rabbit andthe antibody harvested using procedures well known in the art. In oneaspect, such agents can be labeled or used to label peptides of theinvention. In another aspect such agents can be used in diagnostic andscreening methods to monitor agents that may modulate peptide activityor to quantitate the amount of the peptide.

In yet another aspect, the present invention provides an isolatednucleic acid molecule encoding an isolated peptide having or comprisingthe amino acid sequence of TABLE 1 or an analogue, derivative, variant,obvious chemical equivalent thereof. Also provided is a recombinantnucleic acid construct that includes the nucleic acid molecule operablylinked to an expression vector.

In a further aspect, the present invention provides at least one hostcell comprising the recombinant nucleic acid construct of the invention.Also provided is a method for producing a peptide of the invention,e.g., having or comprising the amino acid sequence of TABLE 1 or ananalogue, derivative, variant or obvious chemical equivalent thereof, by(a) culturing the at least one host cell, under conditions allowingexpression of the peptide; and (b) recovering the peptide from the atleast one host cell or culture medium thereof.

In still another aspect, the present invention provides a pharmaceuticalcomposition that includes an isolated peptide having or comprising orconsisting essentially of the amino acid sequence of TABLE 1 or anAnalogue, derivative, variant or obvious chemical equivalent thereof(including a pharmaceutically-acceptable salt, addition salt, or esterof any of the foregoing or polymorph), in combination with apharmaceutically-acceptable carrier, diluent, or excipient.

In another aspect, the present invention provides a method for treatingand/or preventing infection (e.g., a microbial infection) in a subject,by administering to the subject a peptide having or comprising orconsisting essentially of the amino acid sequence, of TABLE 1 or ananalogue, derivative, variant or obvious chemical equivalent thereof orobvious chemical equivalent thereof. By way of example, the subject mayhave, or be at risk of having, infection. In one embodiment, the peptidemodulates innate immunity in the subject, thereby treating and/orpreventing the infection in the subject. The present invention furtherprovides a method for identifying a microbial infection that can betreated with a peptide of the invention. In another aspect, theinvention provides a method for treating or preventing a DPPIV-relatedcondition or disorder.

Exemplary infections which may be treated and/or prevented by the methodof the present invention include an infection by a bacterium (e.g., aGram-positive or Gram-negative bacterium), an infection by a fungus, aninfection by a parasite, and an infection by a virus. In one embodimentof the present invention, the infection is a bacterial infection (e.g.,infection by E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa,Salmonella spp., Staphylococcus aureus, Streptococcus spp., orvancomycin-resistant enterococcus). In another embodiment, the infectionis a fungal infection (e.g., infection by a mould, a yeast, or a higherfungus). In still another embodiment, the infection is a parasiticinfection (e.g., infection by a single-celled or multicellular parasite,including Giardia duodenalis, Cryptosporidium parvum, Cyclosporacayetanensis, and Toxoplasma gondii). In yet another embodiment, theinfection is a viral infection (e.g., infection by a virus associatedwith AIDS, avian flu, chickenpox, cold sores, common cold,gastroenteritis, glandular fever, influenza, measles, mumps,pharyngitis, pneumonia, rubella, SARS, and lower or upper respiratorytract infection (e.g., respiratory syncytial virus)).

In accordance with the method of the present invention, a peptide havingor comprising the amino acid sequence of TABLE 1 or an analogue,derivative, variant or obvious chemical equivalent thereof may beadministered to the subject directly (i.e., by administering the peptideitself) or indirectly (e.g., by administering to the subject a nucleicacid sequence encoding the peptide, in a manner permitting expression ofthe peptide in the subject). The peptide of the invention (or nucleicacid encoding same) may be administered to the subject orally,parenterally (e.g., intradermally, intramuscularly, intraperitoneally,intravenously, or subcutaneously), topically, transdermally,intranasally, by pulmonary administration (e.g., by intratrachealadministration), and/or by osmotic pump.

In yet another aspect, the present invention provides a method forpredicting whether a subject would be responsive to treatment with apeptide comprising the amino acid sequence of TABLE 1 or an analogue,derivative, variant or obvious chemical equivalent thereof, by assayinga diagnostic sample of the subject for DPPIV activity, whereinmodulation, such as reduction of DPPIV activity is indicative that thesubject would be responsive to treatment by the peptide. In one aspect,the subject has or is suspected of having a DPPIV-related condition ordisorder.

Additional aspects and advantages of the present invention will beapparent in view of the description which follows. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described in relation to the drawings, inwhich:

FIGS. 1A, B and C depicts the results of the experiment described inExample 2. % Viability=the amount of bacterial growth relative to thevehicle control (Tris), which is set to 100% bacterial survival, withrespective peptides SEQ. ID. NOs. 1, 5, and 47; Erythr.=erythromycin.

FIG. 2 A-G depicts the results of the experiment described in Example 3.The graph shows colony-forming units per nil (CFU/ml) on the Y-axis, andtreatment group (control=no peptide; SEQ. ID. NOs. 1, 4, 5, 6, 45 and47=treatment with a peptide having the respective amino acid sequence)on the X-axis. The bacterial count of individual mice is shown.

FIGS. 3 A and B depicts the results of the experiment described inExample 4. The graph shows colony-forming units per ml (CFU/ml) on theY-axis, and treatment group (control=no peptide; SEQ. ID. NOs. 1 and5=treatment with a peptide having the respective amino acid sequence) onthe X-axis. The bacterial count of individual mice is shown.

FIG. 4 depicts the results of the human blood infection study describedin Example 5. The graph shows colony-forming units per ml (CFU/ml) onthe Y-axis and treatment group (control=no peptide, SEQ ID No.5=treatment with peptide having the respective amino acid sequence) onthe X-axis.

FIG. 5 depicts the effect of peptide treatment on the ex vivo LPSstimulated cytokine response as described in Example 6.

FIG. 6 depicts the plasma DPPIV dose response curve of SEQ ID No. 5, 51and 83 in human blood as described in Example 8.

FIG. 7 depicts the dose response curve of SEQ. ID. NO. 7 of PCT/CAPCT/CA02/01830, filed Dec. 2, 2002 (KSRIVPAIPVSLL). versus SEQ. ID. No.5 of the present invention. as described in Example 9.

FIGS. 8 A and B depict the enhanced efficacy of antibiotic treatment incombination with SEQ ID NO. 1 and 5 as described in Example 10.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“DPPIV-related disorder” or “DPPIV-related condition” or “DPPIVassociated condition” as used herein means any medical condition thathas been correlated with DPPIV activity and wherein modulation of saidactivity can be used to treat and/or prevent or diagnose said condition.Examples of such conditions include, but are not limited to: HIV/AIDS,autoimmune conditions, such as Rheumatoid Arthritis, multiple sclerosis,cancer (e.g. colon and lung), diabetes, and Graves disease.

“Immune-related disorder” is a condition that is associated with theimmune system of a subject, either through activation or inhibition ofthe immune system, or that can be treated, prevented or diagnosed bytargeting a certain component of the immune response in a subject, suchas the innate immune response.

“Immunologically active” as used herein refers to innate immune activity(e.g. the ability to modulate the innate immune response or componentthereof in a subject) or the ability to modulate DPPIV activity.

“Modulate” or “Modulating” as used herein, for instance such asmodulating DPPIV activity or a particular response, encompasses theincrease or decrease of activity or response in relation to a control orthe normal or baseline level of activity or response under certainconditions. It can also encompass the maintaining of a level of activityor response under conditions that would normally increase or decreasethe level of activity of the peptide. or response.

“Pharmaceutically acceptable salts” refer to the non-toxic alkali metal,alkaline earth metal, and ammonium salts commonly used in thepharmaceutical industry including the sodium, potassium, lithium,calcium, magnesium, barium, ammonium, and protamine zinc salts, whichare prepared by methods well known in the art. The term also includesnon-toxic acid addition salts, which are generally prepared by reactingthe compounds of this invention with a suitable organic or inorganicacid. Representative salts include the chloride, bromide, sulfate,bisulfate, acetate, oxalate, valerate, oleate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, napsylate, trifluoroacetate and the like.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases and which are not biologically or otherwise undesirable, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as trifluoroacetic acid, acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid,succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, menthanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. For a description of pharmaceutically acceptable acid additionsalts as prodrugs, see Bundgaard, H., ed., (1985) Design of Prodrugs,Elsevier Science Publishers, Amsterdam.

“Pharmaceutically acceptable ester” refers to those esters which retain,upon hydrolysis of the ester bond, the biological, effectiveness andproperties of the carboxylic acid or alcohol and are not biologically orotherwise undesirable. For a description of pharmaceutically acceptableesters as prodrugs, see Bundgaard, H., supra. These esters are typicallyformed from the corresponding carboxylic acid and an alcohol. Generally,ester formation can be accomplished via conventional synthetictechniques. (See, e.g., March, Advanced Organic Chemistry, 3rd Ed., JohnWiley & Sons, New York (1985) p. 1157 and references cited therein, andMark et al., Encyclopedia of Chemical Technology, John Wiley & Sons, NewYork (1980).) The alcohol component of the ester will generally comprise(i) a C_(.2)-C_(.12). aliphatic alcohol that can or can not contain oneor more double bonds and can or can not contain branched carbon chainsor (ii) a C_(.7)-C_(.12) aromatic or heteroaromatic alcohols. Thisinvention also contemplates the use of those compositions which are bothesters as described herein and at the same time are the pharmaceuticallyacceptable acid addition salts thereof.

“Pharmaceutically acceptable amide” refers to those amides which retain,upon hydrolysis of the amide bond, the biological effectiveness andproperties of the carboxylic acid or amine and are not biologically orotherwise undesirable. For a description of pharmaceutically acceptableamides as prodrugs, see Bundgaard, H., ed., supra. These amides aretypically formed from the corresponding carboxylic acid and an amine.Generally, amide formation can be accomplished via conventionalsynthetic techniques. (See, e.g., March, Advanced Organic Chemistry, 3rdEd., John Wiley & Sons, New York (1985) p. 1152 and Mark et al.,Encyclopedia of Chemical Technology, John Wiley & Sons, New York(1980).) This invention also contemplates the use of those compositionswhich are both amides as described herein and at the same time are thepharmaceutically acceptable acid addition salts thereof.

“Pharmaceutically or therapeutically acceptable carrier” refers to acarrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredients and which is not toxic tothe host or patient.

“Stereoisomer” refers to a chemical compound having the same molecularweight, chemical composition, and constitution as another, but with theatoms grouped differently. That is, certain identical chemical moietiesare at different orientations in space and, therefore, when pure, hasthe ability to rotate the plane of polarized light. However, some purestereoisomers may have an optical rotation that is so slight that it isundetectable with present instrumentation. The compounds of the instantinvention may have one or more asymmetrical carbon atoms and thereforeinclude various stereoisomers. All immunologically active stereoisomersare included within the scope of the invention.

“Therapeutically or pharmaceutically effective amount” as applied to thecompositions of the instant invention refers to the amount ofcomposition sufficient to induce a desired biological result. Thatresult can be alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forinstance, in the present invention, the result will typically involveenhancement of the innate immune response, reduction of DPPIV activityand/or modulation (such as inhibition or reduction or non-stimulaton) ofthe inflammatory responses to infection or tissue injury.

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is H is or H; Glutamine is Gln or Q; Asparagine is Asnor N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid isGlu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Argor R; and Glycine is Gly or G. In addition, the abbreviation NaI is usedto denote 1-naphthylalanine; Ornithine is Orn or O, Cit is citrulline,Hci is citrulline with one more methylene groups, and Vx or Valine x,wherein the “x” refers to a variation in the backbone of the amino acid,wherein the amino acid linkage is no longer an amide bond, but amethylated amine, this similarly applies to other amino acids with the“x” designation. Also, 2,4-diaminobutyric acid is Dab;2,3-diaminopropionic acid is Dpr or Dapa; N-(4-aminobutyl)-glycine isNlys; hSer is homoserine; Hyp is hydroxyproline; Val(betaOH) ishydroxyvaline; D-Pro is 3,4-dehydroproline; Pyr is pyroglutamine(proline with C═O in ring); Proline with fluorine substitutions on thering; 1,3-thiazolidine-4-carboxylic acid (proline with S in ring); Thiis beta-(2-thienyl)-alanine; Abu is 2-aminobutyric acid; Nva isnorvaline; Nle is norleucine; Hol is homoleucine; and Aib isalpha-aminoisobutyric acid. Pip as used herein refers to(S)-(−)-piperiding-2-carboxylic acid (L-(−)-pipecolic acid; Dhpr is3,4-dehydro-L-proline; Ppm is 2S,4S-4-fluoro-pyrrolidine-2-carboxylicacid (cis-4-fluoro-L-proline); and Thz is R-thiazolidine-4-carboxylicacid (L-thioproline).

In addition to peptides consisting only of naturally-occurring aminoacids, peptidomimetics or peptide analogs are also provided. Peptideanalogs are commonly used in the pharmaceutical industry as non-peptidedrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (Fauchere, J., Adv. Drug Res. 15:29 (1986); Veber andFreidinger, TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229(1987), which are incorporated herein by reference). Peptide mimeticsthat are structurally similar to therapeutically useful peptides may beused to produce an equivalent or enhanced therapeutic or prophylacticeffect. Generally, peptidomimetics are structurally similar to aparadigm peptide (i.e., a peptide that has a biological orpharmacological activity), such as naturally-occurring receptor-bindingpeptide, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of —CH₂NH—, —CH_(2.)S—,—CH₂═CH₂—, —CH|CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—,by methods known in the art and further described in the followingreferences: Spatola, A. F. in Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p.267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,PEPTIDE BACKBONE MODIFICATIONS (general review); Morley, Trends PharmSci (1980) pp. 463 468 (general review); Hudson, D. et al., Int J PeptProt Res 14:177 185 (1979): (—CH₂NH—, CH₂CH₂—); Spatola et al., Life Sci38:1243 1249 (1986): (—CH₂—S); Hann J. Chem. Soc. Perkin Trans. I 307314 (1982): (—CH—CH—, cis and trans); Almquist et al., J Med Chem23:1392 1398 (1980): (—COCH₂—); Jennings-White et al., Tetrahedron Lett23:2533 (1982): (—COCH₂—); Szelke et al., European Application. EP 45665CA: 97:39405 (1982) (—CH(OH)CH_(2.)); Holladay et al., Tetrahedron Lett24:4401 4404 (1983): (—C(OH)CH₂—); and Hruby Life Sci 31:189 199 (1982):(—CH₂—S—); each of which is incorporated herein by reference. In oneaspect, the non-peptide linkage is —CH₂NH—. Such peptide mimetics mayhave significant advantages overpolypeptide embodiments, including, forexample: more economical production, greater chemical stability,enhanced pharmacological properties (half-life, absorption, potency,efficacy, etc.), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, and others. Labeling ofpeptidomimetics usually involves covalent attachment of one or morelabels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) (e.g., immunoglobulinsuperfamily molecules) to which the peptidomimetic binds to produce thetherapeutic effect. Derivatization (e.g., labeling) of peptidomimeticsshould not substantially interfere with the desired biological orpharmacological activity of the peptidomimetic. Generally,peptidomimetics of receptor-binding peptides bind to the receptor withhigh affinity and possess detectable biological activity (i.e., areagonistic or antagonistic to one or more receptor-mediated phenotypicchanges).

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. It isappreciated that those D-amino acid substitutions wherein immunologicalactivity of the peptide is retained are desired.

Description

As described herein, the inventors have identified novel peptides havingand/or comprising the amino acid sequence as shown in TABLE 1 or ananalogue, derivative, variant or obvious chemical equivalent of theamino acid sequences disclosed therein. The inventors have alsodemonstrated that a peptide having or comprising one of the amino acidsequences of TABLE 1 or an analogue, derivative, variant or obviouschemical equivalent thereof and an amidated C-terminus has therapeuticutility in the enhancement of innate immunity. In particular, theinventors have shown that a peptide comprising an amino acid sequence ofTABLE 1 lacked antimicrobial efficacy against S. aureus, yet provided inviva protection in mice infected with S. aureus. The peptide enhancedthe host response to infection, resulting in improved bacterialclearance and host survival. Thus, the novel peptides described can beused as a therapeutic for the treatment of infectious disease. Inanother embodiment, the peptides of the invention have been shown toreduce DPPIV activity, which has been shown to be related to a number ofimmune-related disorders, such as AIDS and HIV disease progression(Blazquez et al. 1992; Vanham et al. 1993; Schols et al. 1998 Oravecz etal. 1995), Graves' disease (Eguchi et al. 1989; Nishikawa et al. 1995),and cancer (Stecca et al. 1997), such as lung and colon cancer, anddiabetes (Hinke et al. 2000; Marguet et al. 2000). Further, DPPIV as anindicator of T-cell activation has been shown to fluctuate in parallelwith several autoimmune diseases such as rheumatoid arthritis (Nakao etal., 1989) and autoimmune thyroiditis (Eguchi et al., 1989). DPPIV hasbeen described as a marker that correlates well with the level ofactivity of these diseases. It has furthermore been studied as anindicator of disease progression in chronic progressive multiplesclerosis (Constantinescu et al., 1995). The peptides of the inventioncan be used in the treatment of such conditions.

Peptides of the Invention

Accordingly, the present invention provides isolated peptides having orcomprising the amino acid sequence of TABLE 1 or an immunologicallyactive analogue, derivative, variant or obvious chemical equivalentthereof. Also provided are pharmaceutically-acceptable salts, acidaddition salts, and esters of the peptides, analogues, derivatives, andvariants of the invention, including those described herein, such asconservative substitution, and, N and C terminus modifications andbackbone modifications, as described herein. Further the peptides of theinvention can be cyclic. As used herein, an “isolated” peptide of theinvention is a peptide which either has no naturally-occurringcounterpart or has been separated or purified from components whichnaturally accompany it. An isolated peptide of the invention can beobtained, for example, by expression of a recombinant nucleic acidencoding the peptide or by chemical synthesis. Because a peptide that ischemically synthesized is, by its nature, separated from the componentsthat naturally accompany it, the synthetic peptide is “isolated”. Assuch, the invention further comprises fusion proteins and peptidescomprising the peptides that differ from the naturally occurringenvironment. Such peptides can include peptides that are metabolizedinto the peptides of the invention.

In one aspect, the isolated peptide of the invention comprises the aminoacid sequence having the formula: “X₁X₂P” (SEQ. ID. NO. 55), wherein Pcan be a proline analogue such as Pip, Thz, Fpro, Dhp, wherein: X₁ isselected from the group consisting of K, R, S, O, or glycine basedcompounds with basic functional groups substituted on the N-terminal(e.g., Nlys), hSer, Val(betaOH), or in another embodiment is selectedfrom the group consisting of K, R, S, and O, or in another embodiment,is R; and wherein X₂ is selected from the group consisting of V, I, R,and W or in one embodiment V, I, and R. In one embodiment X₁ can be Gwhen X₂ is V (e.g SEQ. ID. NO. 88). In another embodiment X₁ can be Kwhen X₂ is H (e.g SEQ. ID. NO. 89) In one embodiment, the isolatedpeptide of the invention is SEQ. ID. NO. 55. In another aspect, it is apeptide of up to 6, 7, 8 or 9 amino acids, or in another embodiment upto 10 amino acids comprising an amino acid sequence of SEQ. ID. NO. 55.In one embodiment, the isolated peptide of SEQ. ID. NO. 55 is SEQ ID NOs83-87. SEQ. ID. NOs. 8, 9, 26, 39, 40, 41, 45, 46 and 48-53, or anisolated peptide of up to 10 amino acids comprising said sequences. Inone embodiment, the peptides are up to 7 amino acids comprising saidsequence, e.g. of SEQ. ID NOs. 62, 8, 9, 13, 26 39, 40, 41, 45, 46, 48,49, 50, 52 and 53. In another embodiment, the isolated peptidecomprising SEQ. ID. NO. 55 is SEQ. ID. NO. 44, which is up to 13 aminoacids.

In another embodiment, the invention provides an isolated peptidecomprising the formula, “X₁X₂X₃P” (SEQ. ID. NO. 56) wherein P can be aproline analogue such as Pip, Thz, Fpro, Dhp, wherein X₁ is selectedfrom the group consisting of K, H, R, S, T, O, or glycine basedcompounds with basic functional groups substituted on the N-terminal(e.g., Nlys), hSer, Val(betaOH), or in another embodiment selected fromthe group consisting of K, H, R and O, or in another embodiment, K, H,R, S, and T, or in another embodiment, K, H, R, S and O, or in anotherembodiment, R, H, K and S, or in another embodiment R, H, and K; andwherein X₂ is selected from the group consisting of A, I, L, V, K, P, G,H, R, S, O, Dab, Dpr, Cit, Hci, Abu, Nva, Nle and where X₂ can beN-methylated, or in another embodiment, selected from the groupconsisting of A, I, L, V, K, P, O, H, and R, where it can beN-methylated; and wherein X₃ is selected from the group consisting of I,V, P, G, H, W, E wherein in one embodiment, X₃ is not N-methylated. Inone embodiment, the isolated peptide can be an amino acid sequence of upto 5, 6, 7, 8, 9 or 10 amino acids, comprising SEQ. ID. NO, 56, or up to5 or 7 amino acids, including SEQ. ID. NOs. 1, 3-7, 10-16, 18, 21-25,27, 28, 31-39, 42, 47, 61, 77, 72, 79, 81, or 90 or an isolated peptideof up to 5, 6, 7, 8, 9, 10, or 11 amino acids comprising SEQ. ID. NO.54. However, in one embodiment when SEQ. ID. NO. 56 is a hexamer, it isselected from the group consisting of SEQ. ID. NOs. 1, 3, 61, 64, or 90,but is not SEQ. ID. NO. 2, or when in one embodiment, when it is apentamer, it is not SEQ. ID. NO. 17. In one embodiment, the isolatedpeptide of the invention does not comprise a peptide comprising SEQ. ID.NOs. 2 or 17. In one embodiment when the peptide is a heptamer it isselected from the group consisting of SEQ. ID. NOs. 18, 32, and 79.

In another embodiment, the invention provides an isolated peptidecomprising the peptide comprising the formula of SEQ. ID. NO. 56 in apentamer or hexamer. In one embodiment, said peptide is immunologicallyactive.

In one embodiment, the isolated peptide of the invention comprises apeptide of formula, “aX₁X₂X₃P” (SEQ. ID. NO. 57) wherein P can be aproline analogue such as Pip, Thz, Fpro, Dhp, wherein X_(I), X₂ and X₃are defined as for SEQ. ID. NO. 56, and wherein “a” is selected from thegroup consisting of S, P, I, R, C, T, L, V, A, G, K, H, R, O, C, M, andF, or in another embodiment is selected from the group consisting of, S,P, I, R, C, T, L, V, A, G, K, H, R, O, C, and M, or in anotherembodiment is selected from the group consisting S, P, I, R, and C, orin another embodiment is S. In one embodiment, the isolated peptidecomprises SEQ. ID. NO. 57, or is a peptide of up to 6, 7, 8, 9, or 10amino acids comprising said sequence. In another embodiment, theisolated peptide is SEQ. ID. NOs. 4, 12, 32, 39, or 47, or an isolatedpeptide up to 7, 8, 9 or 10 amino acids comprising said sequences.

In another embodiment, the isolated peptide of the invention comprises aPeptide of formula, “X₁X₂X₃Pb” (SEQ. ID. NO. 58) wherein P can be aproline analogue such as Pip, Thz, Fpro, Dhp, wherein X₁X₂X₃ are asdefined in SEQ. ID. NO. 56 and “b” is any aliphatic, aromatic, negativeor positively charged amino acid. Or in one embodiment is selected fromthe group consisting of A, A*, E, G, S, L, F, K, W, C, I, V, T, D, Y, R,H, O, Q, N, P, and M, but in one embodiment not P, or in anotherembodiment selected from the group consisting of A, A*, E, H, W, G, S,L, F, and K, or in another embodiment selected from the group consistingof A, A*, G, S, L, K and C, or in one embodiment, selected from thegroup consisting of A, A*, G, S, L, and K. Wherein A* denotes a D aminoacid of Alanine. In one embodiment, when X₁, is R, X₂ is I and b is A,X₃ can be W (e.g. SEQ. ID. NO. 71) In one embodiment, the isolatedpeptide is an amino acid of up to 7, 8, 9 or 10 amino acids comprisingSEQ. ID. NO. 58. In one embodiment, the isolated peptide is or comprisesSEQ. ID. NOs. 5-7, 10, 11, 13-16, 21-25, 27, 28, 31, 33-38 and 42-43. Inanother embodiment, the peptide is of SEQ. ID. NO. 58, wherein “b” isnot P or not RIVPP (SEQ. ID. NO. 17); or where X₃ is not Vx or notRIVxPA.

In one embodiment, the isolated peptide of the invention is or comprisesa peptide similar to SEQ. ID. NO. 58, but wherein X₁ is instead selectedfrom the group consisting of G, GG, or Cit, or wherein “b” is A, X₂ isI, X₃ is V, X₁ is G, GG, or Cit, or the peptide is SEQ. ID. NOs. 19, 20and 36. In one embodiment, the isolated peptide comprises SEQ. ID. NO.31. In another embodiment, the isolated peptide comprises a reversesequence of SEQ. ID. NO. 58, or comprises SEQ. ID. NO. 30.

In one embodiment, the isolated peptide of the invention is or comprisesa peptide having the amino acid sequence of SEQ. ID. NO. 29 (the reversesequence of SEQ. ID. NO. 29).

The peptide of the invention also provides an isolated peptidecomprising the formula, “a₁a₂ X₁X₂X₃P” (SEQ. ID. NO. 59), wherein P canbe a praline analogue such as Pip, Thz, Fpro, Dhp, wherein X₁, X₂ and X₃are as defined in SEQ. ID. NO. 56 and a₁ is selected from the groupconsisting of K, I R, H, O, L, V, A, and G, or in one embodiment, K andI, or in one embodiment K and a₂ is selected from the group consistingof S, P, R T, H, K, O, L, V, A, G, S, and I or in one embodiment, S, P,and R, or in another embodiment, S and P, or in another embodiment P. Inone embodiment, a₁ is not acetylated, or where a₁ is K, K is notacetylated or not SEQ. ID. NO. 2. In one embodiment, the isolatedpeptide is or comprises SEQ. ID. NOs, 1, and 47 or a peptide of up to 10amino acids comprising SEQ. ID. NO. 59.

In another embodiment, the isolated peptide of the invention is orcomprises a peptide of the formula, “a X₁X₂X₃Pb” (SEQ. ID. NO. 60)wherein P can be a proline analogue such as Pip, Thz, Fpro, Dhp, whereinX₁, X₂ and X₃ are as defined in SEQ. ID. NO. 56 and where “a” isselected from the group consisting of S, R, K, H, O, T, I, L, V, A, G orin another embodiment, S, R and I, or in another embodiment S and R, andwherein “b” is selected from the group consisting of A, V, I, L, G, K,H, R, O, S, T, F or in another embodiment, A. In another embodiment, thepeptide of SEQ. ID. NO. 60 is SEQ. ID. NOs. 3, 12 and 39, or a peptideof up to 10 amino acids comprising SEQ. ID. NO. 60 or SEQ. ID. NOs. 3,12 or 39.

As used herein, a “peptide comprising an amino acid sequence of asequence of TABLE 1” or a “peptide comprising an amino acid sequence ofa sequence of TABLE 1” includes the peptide itself, obvious chemicalequivalents thereto, isomers thereof (e.g., isomers, stereoisomers,retro isomers, retro-inverso isomers, all-[D] isomers, all-[L] isomers,or mixed [L] and [D] isomers thereof), conservative substitutionstherein, precursor forms thereof, endoproteolytically-processed formsthereof, such as cleavage of single amino acids from N or C terminals orimmunologically active metabolites of the peptides of the invention,pharmaceutically-acceptable salts and esters thereof, and other formsresulting from post-translational modification. Also included is anyparent sequence, up to and including 10, 9, 8, 7, 6, 5 and 4 amino acidsin length (cyclized, or linear, or branched from the core parentsequence), for which the specified sequence is a subsequence. A personskilled in the art would appreciate that where the peptide in the tableis a trimer, it could be a subsequence of a 10, 9, 8, 7, 6, 5, and 4mer, whereas if the peptide listed in TABLE 1 is a hexamer, it could bea subsequence of a 10, 9, 8, and 7 mer, but not a 5 or 4 mer. Inaddition, the invention comprises sequences that are greater than 10mer, SEQ. ID. NOs. 44 and 54. Further, peptides wherein the majormetabolite are the peptides of Table I are also encompassed within thescope of the present invention. The use of the peptides of the presentinvention include use of peptides wherein the active metabolite is oneor more of the peptides of the present invention. Those modifiedpeptides which retain the immunological activity of the peptides of theinvention are encompassed within the scope of the present invention.These peptides, that are obvious equivalents to and consist essentiallyof SEQ ID NOS:1-90, or SEQ. ID. Nos 1, 3-16, 18-90 or in one embodimentSEQ. ID. Nos 1, 3-16, 18-43, 45-53 or 55-90 are also encompassed withinthe scope of the present invention.

As further used herein, an “obvious chemical equivalent” of a peptide ofthe invention is a molecule which possesses the same desired activity,e.g immunological activity, as peptides described herein, and exhibits atrivial chemical different, or a molecule which is converted, under mildconditions, into a peptide of the invention (e.g., esters, ethers,reduction products, and complexes of the peptides of the invention). Inone embodiment, the invention comprises peptides having the sequencesand motifs of the present invention or peptides comprising same thathave reduced DPPIV activity as compared to a saline control. In oneembodiment, the DDPIV activity is about 75% relative to saline. Inanother embodiment, the DDPIV activity is about 70% relative to saline.Wherein “about” as used herein in relation to DDPIV activity is +/−5%.

Additionally, as used herein, “conservative substitutions” are thoseamino acid substitutions which are functionally equivalent to thesubstituted amino acid residue, either because they have similarpolarity or steric arrangement, or because they belong to the same classas the substituted residue (e.g., hydrophobic, acidic, or basic). Theterm “conservative substitutions”, as defined herein, includessubstitutions having an inconsequential effect on the ability of thepeptide of the invention to enhance innate immunity. Examples ofconservative substitutions include the substitution of a polar(hydrophilic) residue for another (e.g., arginine/lysine,glutamine/asparagine, e/asparagine, or threonine/serine); thesubstitution of a non-polar (hydrophobic) residue (e.g., isoleucine,leucine, methionine, phenylalanine, tyrosine, or valine) for another,the substitution of an acidic residue (e.g., aspartic acid or glutamicacid) for another, or the substitution of a basic residue (e.g.,arginine, histidine, lysine or ornithine) for another.

The term “analogue”, as used herein, includes any peptide having anamino acid sequence substantially identical to a sequence describedherein, in which at least one residue has been conservativelysubstituted with a functionally-similar residue. An “analogue” includesfunctional variants and obvious chemical equivalents of an amino acidsequence of TABLE 1. As further used herein, the term “functionalvariant” refers to the activity of a peptide that demonstrates anability to enhance innate immunity or reduce DPPIV activity, asdescribed herein. An “analogue” includes a variant of an amino acid ofTABLE 1 that has an homologous three-dimensional conformation. An“analogue” further includes any pharmaceutically-acceptable salt of ananalogue as described herein. A “variant” further includes anypharmaceutically-acceptable salt of a variant as described herein.

A “derivative”, as used herein, refers to a peptide of the inventionhaving one or more amino acids chemically derivatized by reaction of afunctional side group. Exemplary derivatized molecules include, withoutlimitation, peptide molecules in which free amino groups have beenderivatized to form salts or amides, by adding acetyl groups, aminehydrochlorides, carbobenzoxy groups, chloroacetyl groups, formyl groups,p-toluene sulfonyl groups, or t-butyloxycarbonyl groups. Free hydroxylgroups may be derivatized to form O-acyl or O-alkyl derivatives.Furthermore, free carboxyl groups may be derivatized to form salts,esters (e.g., methyl and ethyl esters), or hydrazides. Thus, a“derivative” further includes any pharmaceutically-acceptable salt of aderivative as described herein.

In one embodiment of the present invention, the isolated peptide of theinvention has a modified C-terminus and/or a modified N-terminus. Forexample, the isolated peptide may have an amidated C-terminus. Forexample, the amino terminus can be acetylated (Ac) or the carboxyterminus can be amidated (NH₂). However, in one embodiment of theinvention, the peptides of the invention are preferably not acetylatedif such a modification would result in loss of desired immunologicalactivity. Amino terminus modifications include methylating (i.e., —NHCH₃or —NH(CH₃)₂, acetylating, adding a carbobenzoyl group, or blocking theamino terminus with any blocking group containing a carboxylatefunctionality defined by RCOO—, where R is selected from the groupconsisting of naphthyl, acridinyl, steroidyl, and similar groups.Carboxy terminus modifications include replacing the free acid with acarboxamide group or forming a cyclic lactam at the carboxy terminus tointroduce structural constraints.

In one embodiment backbone substitutions can be made, such as NH toNCH₃. The isolated peptide may also be a modification (e.g., a pointmutation, such as an insertion or a deletion, or a truncation) of orcomprising an amino acid sequence of TABLE 1. By way of example, thepeptide may comprise an amino acid sequence of TABLE 1 as modified by atleast one point insertion of a D amino acid as long as desiredimmunological activity is retained. In particular, proline analogs inwhich the ring size of the proline residue is changed from 5 members to4, 6, or 7 members can be employed. Cyclic groups can be saturated orunsaturated, and if unsaturated, can be aromatic or non-aromatic.

One can replace the naturally occurring side chains of the 20genetically encoded amino acids (or D amino acids) with other sidechains with similar properties, for instance with groups such as alkyl,lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide loweralkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and thelower ester derivatives thereof, and with 4-, 5-, 6-, to 7-memberedheterocyclic.

Such substitutions can include but are not necessarily limited to: (1)non-standard positively charged amino acids, like: ornithine, Nlys;N-(4-aminobutyl)-glycine which has the lysine side chain attached to the“N-terminus”. and compounds with aminopropyl or aminoethyl groupsattached to the amino group of glycine. (2), Non-naturally occurringamino acids with no net charge and sidechains similar to arginine, suchas, Cit; citrulline and Hci; citrulline with one more methylene group;(3) non-standard non-naturally occurring amino acids with OH (e.g., likeserine), such as, hSer, homoserine (one more methylen group, Hyp;hydroxyproline, Val(betaOH); hydroxyvaline, Pen; penicillamin,(Val(betaSH); (4) proline derivatives, such as, D-Pro, such as,3,4-dehydroproline, Pyr, pyroglutamine (proline with C═O in ring),Proline with fluorine substitutions on the ring,1,3-thiazolidine-4-carboxylic acid (proline with S in ring); (5)Histidine derivative, such as, Thi; beta-(2thienyl)-alanine; e; or (6)alkyl derivatives, such as, Abu; 2-aminobutyric acid (ethyl group onCalpha), Nva; norvaline (propyl group on Calpha), Me; norleucine (butylgroup on Calpha), Hol; homoleucine (propyl group on Calpha), Aib,alpha-aminoisobutyric acid (valine without methylene group). A personskilled in the art would appreciate that those substitutions that retainthe immunological activity of the parent peptide/sequence.

In another alternative embodiment, the C-terminal carboxyl group or aC-terminal ester can be induced to cyclize by internal displacement ofthe —OH or the ester (—OR) of the carboxyl group or ester respectivelywith the N-terminal amino group to form a cyclic peptide. For example,after synthesis and cleavage to give the peptide acid, the free acid isconverted to an activated ester by an appropriate carboxyl groupactivator such as dicyclohexylcarbodiimide (DCC) in solution, forexample, in methylene chloride (CH₂Cl₂), dimethyl formamide (DMF)mixtures. The cyclic peptide is then formed by internal displacement ofthe activated ester with the N-terminal amine. Internal cyclization asopposed to polymerization can be enhanced by use of very dilutesolutions. Such methods are well known in the art.

One can also cyclize the peptides of the invention, or incorporate adesamino or descarboxy residue at the termini of the peptide, so thatthere is no terminal amino or carboxyl group, to decrease susceptibilityto proteases or to restrict the conformation of the peptide. C-terminalfunctional groups of the compounds of the present invention includeamide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,and carboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

One can also cyclize the peptide by adding an N and/or C terminalcysteine and cyclizing the peptide through disulfide linkages or otherside chain interactions.

One can also incorporate a desamino or descarboxy residue at the terminiof the peptide, so that there is no terminal amino or carboxyl group, todecrease susceptibility to proteases or to restrict the conformation ofthe peptide.

Method of Making Peptides

The present invention contemplates peptides, including peptideanalogues, derivatives, and variants, that are produced synthetically,generated recombinantly, or isolated from native cells. A peptide of theinvention may be synthesized by methods commonly known to one skilled inthe art (e.g., as described in Modern Techniques of Peptide and AminoAcid Analysis (New York: John Wiley & Sons, 1981; and Bodansky, M.,Principles of Peptide Synthesis (New York: Springer-Verlag N.Y., Inc.,1984). Examples of methods that may be employed in the synthesis of thepeptides of the invention include, but are not limited to, solid-phasepeptide synthesis, solution or liquid-method peptide synthesis, andsynthesis using any of the commercially-available peptide synthesizers.In one embodiment, a peptide of the invention is synthesized in vitro,e.g., by chemical means or in vitro translation of mRNA. In anotherembodiment, a peptide of the invention is produced recombinantly, usingconventional techniques and cDNA encoding the peptide. The amino acidsequences of the present invention may further comprise coupling agentsand protecting groups which are used in the synthesis of peptidesequences, and which are well known to one of skill in the art.

Peptide analogues, derivatives, and variants of the invention can bemade by a wide variety of different mutagenesis techniques well known tothose skilled in the art. These techniques can be found in any molecularbiology laboratory manual, including, for example, Sambrook et al.,Molecular Cloning—A Laboratory Manual, 2^(nd) ed. (Plainview, N.Y.: ColdSpring Harbor Press, 1989); or Ausubel et al., Current Protocols inMolecular Biology (John Wiley & Sons). Mutagenesis kits are alsoavailable from many commercial molecular biology suppliers. Methods areavailable to make site-directed, regio-specific, or random mutagenesisin the initial amino acid sequence. After the analogues, derivatives,and variants are produced, they can be screened for the desired abilityto enhance innate immunity, as described herein.

Agents Reactive with Peptide

The present invention further provides an agent reactive with a peptidecomprising an amino acid sequence of TABLE 1 or an analogue, derivative,or variant of thereof. As used herein, “reactive” means the agent hasaffinity for, binds to, or is directed against the peptide of theinvention. As further used herein, an “agent” shall include a protein,polypeptide, peptide, nucleic acid (including DNA or RNA), anon-naturally occurring antibody, Fab fragment, F(ab′)₂ fragment,molecule, compound, antibiotic, drug, and any combination(s) thereof AFab fragment is a univalent antigen-binding fragment of an antibody,which is produced by papain digestion. A F(ab′)₂ fragment is a divalentantigen-binding fragment of an antibody, which is produced by pepsindigestion. Preferably, the agent of the present invention is labeledwith a detectable marker or label. A non-naturally occurring antibodymeans, an antibody that is generated with the peptide associated withanother compound, such as two C-terminal glycine residues and MAPS. MAPSAntigen is attached to the peptide of the present invention via 2glycine residues inserted at the C-terminus of the peptide. Theconstruct can then be administered to an animal, such as a rabbit andthe antibody harvested using procedures well known in the art.

In one embodiment of the present invention, the agent reactive with thepeptide of the invention is an antibody. As used herein, the antibody ofthe present invention may be polyclonal or monoclonal. In addition, theantibody of the present invention may be produced by techniques wellknown to those skilled in the art. Polyclonal antibody, for example, maybe produced by immunizing a mouse, rabbit, or rat with a purifiedpeptide of the invention or a purified peptide linked to an antigen(e.g., MAPS). Monoclonal antibody then may be produced by removing thespleen from the immunized animal, and fusing the spleen cells withmyeloma cells to form a hybridoma which, when grown in culture, willproduce a monoclonal antibody. See, e.g., J. G. R. Hurrel, MonoclonalHybridoma Antibodies: Techniques and Applications (Boco Raton, Fla.: CRCPress Inc., 1982).

The antibody and even the peptides themselves of the invention may belabeled with a detectable marker or label. Labeling of an antibody orpeptide may be accomplished using one of a variety of labelingtechniques, including peroxidase, chemiluminescent labels known in theart, and radioactive labels known in the art. The detectable marker orlabel of the present invention may be, for example, a nonradioactive orfluorescent marker, such as biotin, fluorescein (FITC), acridine,cholesterol, or carboxy-X-rhodamine, which can be detected usingfluorescence and other imaging techniques readily known in the art.Alternatively, the detectable marker or label may be a radioactivemarker, including, for example, a radioisotope. The radioisotope may beany isotope that emits detectable radiation, such as ³⁵S, ³²P, ¹²⁵I, ³H,or ¹⁴C. Radioactivity emitted by the radioisotope can be detected bytechniques well known in the art. For example, gamma emission from theradioisotope may be detected using gamma imaging techniques,particularly scintigraphic imaging. Preferably, the agent of the presentinvention is a high-affinity antibody labeled with a detectable markeror label.

Isolated Nucleic Acid Molecules

In addition, the present invention provides an isolated nucleic acidmolecule encoding a peptide comprising an amino acid sequence of TABLE 1or an analogue, derivative, variant or obvious chemical equivalentthereof, including a conjugated peptide (e.g. a carrier-peptideconstruct) or other peptide, or a pro-peptide that metabolizes orcleaves to an immunologically active peptide of TABLE 1. Due to thedegeneracy of the genetic code, the nucleic acid molecule of theinvention includes a multitude of nucleic acid substitutions that willalso encode a peptide of the invention. The present invention furtherprovides a nucleic acid which hybridizes to the isolated nucleic acidmolecule encoding an amino acid sequence of TABLE 1 or an analogue,derivative, or variant thereof.

The nucleic acid molecules of the present invention may be DNA or RNA.They may be prepared by a variety of techniques known to those skilledin the art, including, without limitation, automated synthesis ofoligonucleotides using commercially-available oligonucleotidesynthesizers, such as the Applied Biosystems Model 392 DNA/RNAsynthesizer. In addition, the nucleic acid molecules of the presentinvention may be labeled with one or more detectable markers or labels.Labeling of the nucleic acid molecules may be accomplished using one ofa number of methods known in the art—e.g., nick translation, endlabeling, fill-in end labeling, polynucleotide kinase exchange reaction,random priming, or SP6 polymerase (for riboprobe preparation)—along withone of a variety of labels—e.g., radioactive labels, such as ³⁵S, ³²P,or ³H, or nonradioactive labels, such as biotin, fluorescein (FITC),acridine, cholesterol, or carboxy-X-rhodamine (ROX).

The present invention also provides a recombinant nucleic acid constructcomprising a nucleic acid molecule of the invention operably linked toan expression vector. As used herein, an “expression vector” is a DNAconstruct containing a DNA sequence which is operably linked to asuitable control sequence capable of effecting the expression of the DNAin a suitable host. The vector may be, for example, a plasmid, a phageparticle, or a potential genomic insert. As further used herein, theterm “operably linked” describes a functional relationship between twoDNA regions. Expression vectors suitable for use in the presentinvention comprise at least one expression control element (e.g.,operator, promoter, lac system, leader sequence, termination codon,and/or polyadenylation signal) operably linked to the nucleic acidmolecule encoding a peptide of the invention. In one embodiment, theexpression vector is a eukaryotic expression vector that functions ineukaryotic cells (e.g., a retroviral vector, a vaccinia virus vector, anadenovirus vector, a herpes virus vector, or a fowl pox virus vector).

Once operably linked to a nucleic acid molecule of the invention, theexpression vector may be introduced into a recipient cell by any in vivoor ex vivo means suitable for transfer of nucleic acid, including,without limitation, electroporation, DEAR Dextran transfection, calciumphosphate transfection, lipofection, monocationic liposome fusion,polycationic liposome fusion, protoplast fusion, creation of an in vivoelectrical field, DNA-coated microprojectile bombardment, injection withrecombinant replication-defective viruses, homologous recombination,viral vectors, naked DNA transfer, or any combination thereof.Recombinant viral vectors suitable for transfer of nucleic acid include,but are not limited to, vectors derived from the genomes of viruses suchas retrovirus, HSV, adenovirus, adeno-associated virus, Semiliki Forestvirus, cytomegalovirus, and vaccinia virus.

The present invention further provides at least one host cell comprisingthe recombinant nucleic acid construct of the invention. The host cellof the invention is transformed with the nucleic acid constructdescribed herein. The host cell may be eukaryotic (e.g., an animal,plant, insect, or yeast cell) or prokaryotic (e.g., E. coli).

In addition, the present invention provides a method for producing apeptide comprising an amino acid sequence of TABLE 1 or an analogue,derivative, or variant thereof. The method comprises the steps of: (a)culturing at least one host cell comprising a recombinant nucleic acidconstruct, as described herein, under conditions allowing expression ofthe peptide; and (b) recovering the peptide from the at least one hostcell or from the culture medium thereof. The recombinant peptide can berecovered as a crude lysate; it can also be purified by standard proteinpurification procedures known in the art, including, without limitation,affinity and immunoffinity chromatography, differential precipitation,gel electrophoresis, ion-exchange chromatography, isoelectric focusing,size-exclusion chromatography, and the like.

Pharmaceutical Composition

The present invention further provides a pharmaceutical compositioncomprising a peptide comprising an amino acid sequence of the invention,e.g. TABLE 1 (SEQ ID NOS:1-90) or SEQ. ID. NOs. 1, 3-16, or 18-90, or apeptide comprising said peptide of SEQ ID NO: 1, 3-16, 18-43, 45-53 or55-90 of up to 7, 8, 9 or 10 amino acids as the case may be, or ananalogue, derivative, or variant thereof (which includes apharmaceutically-acceptable salt, acid addition salt or ester of any ofthe foregoing), in combination with at least onepharmaceutically-acceptable carrier, diluent, or excipient. Thepharmaceutically-acceptable carrier, diluent, or excipient must be“acceptable” in the sense of being compatible with the other ingredientsof the composition, and not deleterious to the recipient thereof.Examples of acceptable pharmaceutical carriers, diluents, and excipientsinclude, without limitation, carboxymethyl cellulose, crystallinecellulose, glycerin, gum arabic, lactose, magnesium stearate, methylcellulose, powders, saline, sodium alginate, sucrose, starch, talc, andwater, among others. Formulations of the pharmaceutical composition ofthe invention, as described herein, may be conveniently presented inunit dosage.

Uses

The peptides of the invention have been shown to have therapeuticutility in enhancing innate immunity. The enhancement of innate immunityis demonstrated by the lack of antimicrobial activity (Example 2) andthe protection against infection in in vivo models (Examples 3-6 and 10)and also by the DPPIV assays of Examples 7, 8 and 9. Accordingly, thepresent invention also provides a method for treating and/or preventinginfection in a subject. As used herein, the “subject” is a bird (e.g., achicken, turkey, etc.) or a mamma) (e.g., a cow, dog, human, monkey,mouse, pig, rat, etc.). In one embodiment, the subject is a human. Thesubject may have, or be at risk of having, an infection. By way ofexample, the infection may be a microbial infection. Microbialinfections which may be treated by the method of the present inventioninclude, without limitation, infection by a bacterium, infection by afungus, infection by a parasite, and infection by a virus.

Most bacterial pathogens are present in the general environment, or inthe hogs normal bacterial flora. Bacteria have evolved the ability tocause severe disease by acquiring different mechanisms (called virulencefactors) which enable them to colonize, disseminate within, and invadehost tissues. When these pathogenicity factors are suppressed, bacteriaare no longer able to maintain themselves in host tissues, and,therefore, cannot cause disease. Exemplary bacteria which may be treatedby the method of the present invention include, without limitation, E.coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella spp.(e.g., Salmonella typhimurium), Staphylococcus aureus, Streptococcusspp., and vancomycin-resistant enterococcus.

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that isnoted for its environmental versatility, its ability to cause disease insusceptible individuals, and its resistance to antibiotics. It is aversatile organism that grows in soil, marshes, and coastal marinehabitats, and on plant and animal tissues. The most serious complicationof cystic fibrosis is respiratory tract infection by P. aeruginosa.Cancer and burn patients also commonly suffer serious infections by thisorganism, as do certain other individuals with immune systemdeficiencies. Unlike many environmental bacteria, P. aeruginosa has aremarkable capacity to cause disease in susceptible hosts.

Staphylococcus aureus is a Gram-positive spherical bacterium, about 1micrometer in diameter, that thrives in microscopic clusters. It is oneof the most important human pathogens, causing both community-acquiredand nosocomial infections that range from endocarditis to pneumonia.Although S. aureus is generally classified as an extracellular pathogen,recent data have revealed its ability to infect various types of hostcells, e.g., both professional phagocytes and non-phagocytes, includingendothelial cells, fibroblasts, and others. This invasion is initiatedby the adherence of S. aureus to the cell surface, a process in whichstaphylococcal fibronectin-binding proteins play a prominent role.Phagocytosed S. aureus can either induce apoptosis of the host cell orsurvive for several days in the cytoplasm—which is thought to be devoidof anti-staphylococcal effector mechanisms.

S. aureus colonizes nasal passages, skin surfaces, mucous membranes, andareas around the mouth, genitals, and rectum. S. aureus may causesuperficial skin lesions, such as boils, styes, and furuncles. Moreserious infections include pneumonia, mastitis, phlebitis, meningitis,and urinary tract infections; deep-seated infections includeosteomyelitis and endocarditis.

Exemplary fungi which may be treated by the method of the presentinvention include, without limitation, moulds, yeasts, and higher fungi.All fungi are eukaryotic, and have sterols, but not peptidoglycan, intheir cell membranes. Fungal infections, or mycoses, are classifiedaccording to the degree of tissue involvement and the mode of entry intothe host. In the immunocompromised host, a variety of non-pathogenicfungi, or fungi that are normally mild, can cause potentially fatalinfections.

Parasites are organisms that derive nourishment and protection fromother living organisms (known as hosts). They may be transmitted fromanimals to humans, from humans to humans, or from humans to prima's.Several parasites have emerged as significant causes of food-borne andwater-borne disease. They may be transmitted from host to host throughconsumption of contaminated food and water, or through ingestion of asubstance that has come into contact with the stool (feces) of aninfected person or animal. Parasites live and reproduce within thetissues and organs of infected human and animal hosts, and are oftenexcreted in feces. There are different types of parasites, ranging insize from tiny, single-celled, microscopic organisms (protozoa), tolarger, multi-cellular worms (helminths) that may be seen without amicroscope. Examples of common parasites which may be treated by themethod of the present invention include, without limitation, Giardiaduodenalis, Cryptosporidium parvum, Cyclospora cayetanensis, andToxoplasma gondii.

Viruses are unlike fungi and bacteria, lacking many of the attributes offree-living cells. A single virus particle is a static structure, quitestable and unable to change or replace its parts. Only when associatedwith a cell does a virus become capable of replicating and acquiringsome of the attributes of a living system. Viruses cause numerousdiseases, including such upper respiratory tract infections (URTIs) asthe common cold and pharyngitis (sore throat). Other examples of viruseswhich may be treated by the method of the present invention include,without limitation, viruses associated with AIDS, avian flu, chickenpox,cold sores, common cold, gastroenteritis (especially in children),glandular fever, influenza, measles, mumps, pharyngitis, pneumonia,rubella, SARS, and lower respiratory tract infection (e.g., respiratorysyncytial virus, or RSV)).

The inventors have demonstrated herein that peptides comprising orconsisting essentially of the amino acid sequence of TABLE 1 or SEQ ID.NO. 1, 3-16, or 18-90 or in another embodiment, SEQ. ID. NO. 1, 3-16,18-43, 45-53, or 55-90, or analogue, derivative, variant or obviouschemical equivalent thereof have efficacy in the prevention and/ortreatment of infection. Accordingly, the present method of treatingand/or preventing infection in a subject comprises administering to thesubject a peptide comprising the amino acid sequence of TABLE 1 or SEQ.ID. NO. 1, 3-16, 18-90, or analogue, derivative, or variant thereof orobvious chemical equivalent thereof. It is within the confines of thepresent invention that the peptide of the invention may be linked toanother agent or administered in combination with another agent, such asan antibiotic (e.g., penicillin, methicillin, or vancomycin), in orderto increase the effectiveness of the treatment and/or prevention ofinfection, and/or increase the efficacy of targeting.

In one embodiment of the present invention, the peptide of the inventioncomprises the amino acid sequence of TABLE 1 or SEQ. ID. NO. 1, 3-16,18-90, or in another embodiment, SEQ. ID. NO. 1, 3-16, 18-43, 45-53, or55-90, or analogue, derivative, or variant thereof or obvious chemicalequivalent thereof. In another embodiment, the peptide of the inventionmodulates innate immunity in the subject, thereby treating and/orpreventing the infection in the subject. The innate immune response isthe front line response to a pathogen encounter. It comprises amultiplicity of mechanisms to prevent development of infectious disease.One such mechanism involves the priming and recruitment of immuneeffector cells.

In one embodiment, the peptides of the invention can enhance innateimmunity or the innate immune response, while limiting inflammation.

In another embodiment, the peptides of the invention have been shown tobe modulators of DPPIV activity. They have been shown to reduce DPPIVactivity. As such, they would be useful in the screening of subjects whomay benefit from administration of the peptides to treat a particularimmunological condition, comprising taking a sample from a subjectsuspected or known to have a DPPIV-related condition, incubating ittogether with a peptide of the invention and a DDPIV substrate and thenmonitoring the effect of the peptide on DDPIV activity in comparison toa control wherein a reduction in activity would indicate the potentialbenefit of administration of the peptide to the subject to treat aDPPIV-related condition. In another embodiment modulation of DPPIVactivity (e.g decrease in DPPIV activity) in the presence of the peptideas compared to the control can be indicative of a DPPIV-relatedcondition. As such, the peptides of the invention can be used in thediagnosis of DPPIV-related conditions. In another aspect the peptides ofthe invention would be useful in the treatment of a number ofimmunological disorders, such as DPPIV-related disorder, such as:HIV/AIDS, Grave's disease, cancer (such as lung and colon cancer),diabetes, and autoimmune disorders such as rheumatoid arthritis andmultiple sclerosis.

Administration

In accordance with the method of the present invention, a peptide of thepresent invention as described herein may be administered to the subjectdirectly, in an amount effective to treat and/or prevent infection inthe subject and/or to treat or prevent a DPPIV-related condition, e.g. atherapeutic effective amount. Similarly, a peptide as described hereinmay be administered to the subject indirectly, by administering to thesubject a nucleic acid sequence encoding the peptide, in a mannerpermitting expression of the peptide in the subject, and in an amounteffective to treat and/or prevent infection.

Furthermore, a peptide of the invention, or a nucleic acid moleculeencoding same, may be administered to a subject in an amount effectiveto treat the infection or inflammation or a DPPIV or innateimmunity-related condition in the subject. As used herein, the phrase“effective to treat the infection or inflammation or DPPIV or an innateimmunity-related condition” means effective to ameliorate or minimizethe clinical impairment or symptoms resulting from infection (by abacterium, fungus, parasite, virus, etc.) and the attendantinflammation. For example, where the subject is infected with a microbe,the amount of peptide (or nucleic acid encoding same) which is effectiveto treat the microbial infection is that which can ameliorate orminimize the symptoms of the microbial infection, including, withoutlimitation, headache, stiff neck, anorexia, nausea, vomiting, diarrhea,abdominal discomfort, acute renal failure, changing manifestations ofischemic damage to multiple organs, fever, and thrombocytopenia. Theamount of peptide (or nucleic acid encoding same) effective to treat aninfection or inflammation in a subject will vary depending on theparticular factors of each case, including the subject's weight and theseverity of the subject's condition. The appropriate amount of peptide(or nucleic acid encoding same) can be readily determined by the skilledartisan. Similarly the amount effective to treat a DPPIV-relatedcondition can vary depending on a number of similar factors known to aperson skilled in the art.

Similarly, in the method of the present invention, a peptide of theinvention, or a nucleic acid molecule encoding same, may also beadministered to a subject at risk of developing an infection,inflammation or DPPIV or an innate immunity-related condition, in anamount effective to prevent the infection, inflammation or DPPIV or aninnate immunity-related condition in the subject. As used herein, thephrase “effective to prevent the infection, inflammation or DPPIV or aninnate immunity-related condition” includes effective to hinder orprevent the development or manifestation of clinical impairment orsymptoms resulting from infection (by a bacterium, fungus, parasite,virus, etc.), inflammation or DPPIV or an innate immunity-relatedcondition. The amount of peptide (or nucleic acid encoding same)effective to prevent an infection in a subject will vary depending onthe particular factors of each case, including the subject's sex, weightand the severity of the subject's condition, nature of condition, siteof infection, and mode of administration. The appropriate amount ofpeptide (or nucleic acid encoding same) can be readily determined by theskilled artisan.

In one embodiment the invention includes administration of the peptidewith an antibiotic, e.g. vancomycin to treat an infection. In this casethe antibiotic can be administered at the same time in separateformulations or within one formulation or pharmaceutical preparation.Alternatively, the antibiotic can be administered before or afteradministration of the peptide of the invention. Pharmaceuticalpreparation comprising both an antibiotic and a peptide of the inventionand a pharmaceutical acceptable carrier are contemplated to beencompassed within the present invention.

The peptide of the invention, or the nucleic acid sequence encodingsame, as disclosed herein, may be administered to a human or animalsubject by known procedures, including, without limitation, oraladministration, parenteral administration (e.g., epifascial,intracapsular, intracutaneous, intradermal, intramuscular, intraorbital,intraperitoneal, intraspinal, intrasternal, intravascular, intravenous,parenchymatous, or subcutaneous administration), topical administration,transdermal administration, intranasal administration, pulmonaryadministration (e.g., intratracheal administration), and administrationby osmotic pump. In one embodiment, the method of administration isparenteral administration, by intravenous or subcutaneous injection.

When applied topically, the peptides of the invention are suitablycombined with other ingredients, such as carriers and/or adjuvants orpenetration enhancers known in the art. There are no limitations on thenature of such other ingredients, except that they must bepharmaceutically acceptable and efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the composition. In one embodiment, they are notirritable to the skin or mucosal membrane to which they are applied.Examples of suitable vehicles include ointments, creams, gels, orsuspensions, including colloids, with or without purified collagen. Thecompositions also may be impregnated into transdermal patches, plasters,and bandages, preferably in liquid or semi-liquid form.

For oral administration, the formulation of the peptide (or nucleic acidencoding same) may be presented as capsules, tablets, powders, granules,or as a suspension or liquid. The formulation may have conventionaladditives, such as lactose, mannitol, corn starch, or potato starch. Theformulation also may be presented with binders, such as crystallinecellulose, cellulose derivatives, acacia, corn starch, or gelatins.Additionally, the formulation may be presented with disintegrators, suchas corn starch, potato starch, or sodium carboxymethylcellulose. Theformulation may be further presented with dibasic calcium phosphateanhydrous or sodium starch glycolate. Finally, the formulation may bepresented with lubricants, such as talc or magnesium stearate.

For parenteral administration, the peptide (or nucleic acid encodingsame) may be combined with a sterile aqueous solution, which ispreferably isotonic with the blood of the subject. Such a formulationmay be prepared by dissolving a solid active ingredient in watercontaining physiologically-compatible substances, such as sodiumchloride, glycine, and the like, and having a buffered pH compatiblewith physiological conditions, so as to produce an aqueous solution,then rendering said solution sterile. The formulation may be presentedin unit or multi-dose containers, such as sealed ampoules or vials. Theformulation also may be delivered by any mode of injection, includingany of those described herein.

For transdermal administration, the peptide (or nucleic acid encodingsame) may be combined with skin penetration enhancers, such as propyleneglycol, polyethylene glycol, isopropanol, ethanol, oleic acid,N-methylpyrrolidone, and the like, which increase the permeability ofthe skin to the peptide or nucleic acid, and permit the peptide ornucleic acid to penetrate through the skin and into the bloodstream. Thecomposition of enhancer and peptide or nucleic acid also may be furthercombined with a polymeric substance, such as ethylcellulose,hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone,and the like, to provide the composition in gel form, which may bedissolved in solvent, such as methylene chloride, evaporated to thedesired viscosity, and then applied to backing material to provide apatch. The peptide or nucleic acid may be administered transdermally, ator near the site on the subject where the infection may be localized.Alternatively, the peptide or nucleic acid may be administeredtransdermally at a site other than the affected area, in order toachieve systemic administration.

For intranasal administration (e.g., nasal sprays) and/or pulmonaryadministration (administration by inhalation), formulations of thepeptide or nucleic acid, including aerosol formulations, may be preparedin accordance with procedures well known to persons of skill in the art.Aerosol formulations may comprise either solid particles or solutions(aqueous or non-aqueous). Nebulizers (e.g., jet nebulizers, ultrasonicnebulizers, etc.) and atomizers may be used to produce aerosols fromsolutions (e.g., using a solvent such as ethanol); metered-dose inhalersand dry-powder inhalers may be used to generate small-particle aerosols.The desired aerosol particle size can be obtained by employing any oneof a number of methods known in the art, including, without limitation,jet-milling, spray drying, and critical-point condensation.

Pharmaceutical compositions for intranasal administration may be solidformulations (e.g., a coarse powder), and may contain excipients (e.g.,lactose). Solid formulations may be administered from a container ofpowder held up to the nose, using rapid inhalation through the nasalpassages. Compositions for intranasal administration may also compriseaqueous or oily solutions of nasal spray or nasal drops. For use with asprayer, the formulation of peptide or nucleic acid may comprise anaqueous solution and additional agents, including, for example, anexcipient, a buffer, an isotonicity agent, a preservative, or asurfactant. A nasal spray may be produced, for example, by forcing asuspension or solution of the peptide or nucleic acid through a nozzleunder pressure.

Formulations of the peptide or nucleic acid for pulmonary administrationmay be presented in a form suitable for delivery by an inhalationdevice, and may have a particle size effective for reaching the lowerairways of the lungs or sinuses. For absorption through mucosalsurfaces, including the pulmonary mucosa, the formulation of the presentinvention may comprise an emulsion that includes, for example, abioactive peptide, a plurality of submicron particles, a mucoadhesivemacromolecule, and/or an aqueous continuous phase. Absorption throughmucosal surfaces may be achieved through mucoadhesion of the emulsionparticles.

Pharmaceutical compositions for use with a metered-dose inhaler devicemay include a finely-divided powder containing the peptide or nucleicacid as a suspension in a non-aqueous medium. For example, the peptideor nucleic acid may be suspended in a propellant with the aid of asurfactant (e.g., sorbitan trioleate, soya lecithin, or oleic acid).Metered-dose inhalers typically use a propellent gas (e.g., achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon) stored in a container (e.g., a cannister) as a mixture(e.g., as a liquefied, compressed gas). Inhalers require actuationduring inspiration. For example, actuation of a metering valve mayrelease the mixture as an aerosol. Dry-powder inhalers usebreath-actuation of a mixed powder.

The peptide or nucleic acid of the present invention also may bereleased or delivered from an osmotic mini-pump or other timed-releasedevice. The release rate from an elementary osmotic mini-pump may bemodulated with a microporous, fast-response gel disposed in the releaseorifice. An osmotic mini-pump would be useful for controlling release,or targeting delivery, of the peptide or nucleic acid.

In accordance with methods described herein, the peptide of theinvention may be administered to a subject by introducing to the subjectthe peptide itself, or by introducing to the subject a nucleic acidencoding the peptide in a manner permitting expression of the peptide.Accordingly, in one embodiment of the present invention, infection in asubject may be treated or prevented by administering to the subject anamount of a peptide of the invention. In a further embodiment of thepresent invention, infection in the subject may be treated or preventedby administering to the subject a nucleic acid sequence encoding apeptide of the invention, in a manner permitting expression of thepeptide in the subject.

The peptides of the present invention may be administered or introducedto a subject by known techniques used for the introduction of proteinsand other drugs, including, for example, injection and transfusion.Where an infection is localized to a particular portion of the body ofthe subject, it may be desirable to introduce the therapeutic peptidedirectly to that area by injection or by some other means (e.g., byintroducing the peptide into the blood or another body fluid). Theamount of peptide to be used is an amount effective to treat and/orprevent the infection in the subject, as defined above, and may bereadily determined by the skilled artisan.

In the method of the present invention, the peptide also may beadministered or introduced to the subject by introducing into asufficient number of cells of the subject a nucleic acid encoding thepeptide, in a manner permitting expression of the peptide. The amount ofnucleic acid encoding the therapeutic peptide is an amount that willproduce the peptide in an amount effective to treat and/or preventinfection, as defined above, in the subject. This amount may be readilydetermined by the skilled artisan.

Nucleic acid encoding the peptide of the present invention may beintroduced to the subject using conventional procedures known in theart, including, without limitation, electroporation, DEAE Dextrantransfection, calcium phosphate transfection, lipofection, monocationicliposome fusion, polycationic liposome fusion, protoplast fusion,creation of an in vivo electrical field, DNA-coated microprojectilebombardment, injection with recombinant replication-defective viruses,homologous recombination, in vivo gene therapy, ex vivo gene therapy,viral vectors, naked DNA transfer, or any combination thereof.Recombinant viral vectors suitable for gene therapy include, but are notlimited to, vectors derived from the genomes of viruses such asretrovirus, HSV, adenovirus, adeno-associated virus, Semiliki Forestvirus, cytomegalovirus, and vaccinia virus.

It is also within the confines of the present invention that a nucleicacid encoding a peptide of the invention may be introduced into suitablecells in vitro, using conventional procedures, to achieve expression ofthe therapeutic peptide in the cells. Cells expressing the peptide thenmay be introduced into a subject to treat and/or prevent infection invivo. In such an ex vivo gene therapy approach, the cells are preferablyremoved from the subject, subjected to DNA techniques to incorporatenucleic acid encoding the therapeutic peptide, and then reintroducedinto the subject.

It is also within the confines of the present invention that aformulation containing a peptide of the invention, or a nucleic acidencoding same, may be further associated with apharmaceutically-acceptable carrier, diluent, or excipient, therebycomprising a pharmaceutical composition. Pharmaceutical compositions ofthe invention, and exemplary carriers, diluents, and excipients, aredescribed above.

The formulations of the present invention may be prepared by methodswell-known in the pharmaceutical arts. For example, the peptide of theinvention, or a nucleic acid encoding same, may be brought intoassociation with a carrier, diluent, or excipient, as a suspension orsolution. Optionally, one or more accessory ingredients (e.g., buffers,flavoring agents, surface active agents, and the like) also may beadded. The choice of carrier will depend upon the route ofadministration. The pharmaceutical composition would be useful foradministering the peptide of the present invention, or a nucleic acidmolecule encoding same, to a subject, in order to treat and/or preventinfection. The peptide or nucleic acid is provided in an amount that iseffective to treat and/or prevent infection in a subject to whom thepharmaceutical composition is administered. This amount may be readilydetermined by the skilled artisan, as described above.

Diagnostics and Screening Assays

The present invention provides a method for diagnosing a subject who issuspected of having an innate immune condition, or DPPIV-relatedcondition, for predicting whether a subject would be responsive totreatment with a peptide of the invention, such as a peptide comprisingthose listed in TABLE 1, or SEQ ID. NO. 1, 3-16, or 18-90 or in anotherembodiment, SEQ. ID. NO. 1, 3-16, 18-43, 45-53, or 55-90, or ananalogue, derivative, variant or obvious chemical equivalent thereof,and to screening for agents that would modulate (e.g., enhance, inhibitor mimic) the immunological effect of the peptides of the invention. Inanother embodiment, the invention provides methods for screening forimmunologically active analogues, derivatives, and variants of thepeptides of the invention or those listed in TABLE 1 or toimmunologically active modifications thereof.

In one embodiment, a method for predicting whether a patient with aimmunological disorder, such as an innate immune-related condition wouldbe responsive to treatment with a peptide of the invention comprisesobtaining a biological sample from the subject, administering a peptideof the invention to said sample, and monitoring levels of apredetermined marker that is indicative of the condition, such as DPPIVfor a DPPIV-related condition, an inflammatory biomarker for aninfection, cell viability or bacterial load, in comparison to a positiveand/or negative control. The positive control can be a sample from asubject with a known immunological condition. A negative control can bea sample from the same subject that is not administered the peptide. Ifthe peptide modulates the activity, level of marker, or cell viabilityin relation to the control, the subject may have such immunologicaldisorder and may benefit from treatment with the peptide.

More particularly, in one aspect of the invention, if the subject has oris suspected of having a DPPIV-related condition, then monitoring DPPIVactivity as the marker for the condition would be appropriate. In oneaspect, reduction of DPPIV activity in comparison to the control wouldbe indicative that the subject would be responsive to treatment with thepeptide. Alternatively, if the subject has or was suspected of having aninfection, then obtaining a sample from the patient, monitoring it forpathogen load, cell viability or cytokine expression or productionpattern in comparison to a sample from the patient after administrationof the peptide, wherein pathogen load is less or cell viability ishigher or cytokine expression/production is altered in the patient afteradministration of the peptide, is indicative that the subject wouldbenefit from peptide treatment or has an immunological disorder.

In another embodiment, if one wishes to see whether a peptide ormodification of a peptide of TABLE 1 or other agent would have the sameimmunological activity as the peptide of the invention, one can monitorthe effect of the peptide on DPPIV activity in comparison to thereference peptide with known modulatory affects, on a sample (eitherfrom a mouse infected with an agent or a known DPPIV-related condition),or to monitor prevention, administration of the peptide or agent to asample, inducing said infection or DPPIV-related condition in saidsample and then monitoring whether the peptide modulated or inhibiteddevelopment of said infection or DPPIV-related condition, or immuneresponse. Said sample can be an animal model, wherein induction of thecondition or infection is done in an accepted animal model in accordancewith ethical guidelines and then the animal or appropriate biologicalsample of the animal is screened for effect of the peptide.

The present invention further provides a method for predicting whether asubject would be responsive to treatment for a microbial infectionwherein the treatment comprises administering to the subject a peptidecomprising an amino acid sequence of the invention, such as of TABLE 1or SEQ ID. NO. 1, 3-16, or 18-90 or SEQ. M. NO. 1, 3-16, 18-43, 45-53,or 55-90, or an analogue, derivative, variant or obvious chemicalequivalent thereof. The method includes assaying a diagnostic sample ofthe subject for one or more biomarkers (such as an inflammatorybiomarker), wherein the presence of at least one biomarker (such as aninflammatory biomarker) is indicative that the subject would beresponsive to the treatment.

As used herein a “biomarker” or “marker is any suitable biomarker knownto be, or recognized as being, related to the condition (e.g. immunecondition, infection, inflammatory condition, DPPIV-related condition,innate immune condition), and includes any molecule derived from a gene(e.g., a transcript of the gene), a sense (coding) or antisense(non-coding) probe sequence derived from a gene, or a partial-length orfull-length translation product of a gene, or an antibody thereto, whichcan be used to monitor a condition, disorder, or disease associated withthe immune response, innate immune response, inflammation, and/or aDPPIV-related condition.

According to the method of the present invention, the diagnostic sampleof a subject may be assayed in vitro or in vivo. Where the assay isperformed in vitro, a diagnostic sample from the subject may be removedusing standard procedures. The diagnostic sample may be tissue,including any muscle tissue, skin tissue, or soft tissue, which may beremoved by standard biopsy. In addition, the diagnostic sample may be abodily fluid, including blood, saliva, serum, or urine. The subject orpatient may be known to have a microbial infection or otherimmunological disorder such as a DPPIV-related condition, suspected ofhaving a microbial infection or other immunological condition, such asan innate immune condition or DPPIV-related condition, or believed notto have a microbial infection or other immunological condition, such asan innate-immune condition, or DPPIV-related condition.

In accordance with the method of the present invention, a diagnosticsample of the subject may be assayed for expression of one or moredesired markers. As used herein, “expression” means the transcription ofan inflammatory-marker gene into at least one mRNA transcript, or thetranslation of at least one mRNA into a marker protein. Accordingly, adiagnostic sample may be assayed for marker expression by assaying for amarker protein, marker cDNA, or marker mRNA. The appropriate form of themarker will be apparent based on the particular techniques discussedherein.

Protein to be assayed may be isolated and purified from the diagnosticsample of the subject or patient using standard methods known in theart, including, without limitation, extraction from a tissue (e.g., witha detergent that solubilizes the protein) where necessary, followed byaffinity purification on a column, chromatography (e.g., FPLC and HPLC),immunoprecipitation (with an antibody to an inflammatory marker ofinterest), and precipitation (e.g., with isopropanol and a reagent suchas Trizol). Isolation and purification of the protein may be followed byelectrophoresis (e.g., on an SDS polyacrylamide gel). It is contemplatedthat the diagnostic sample may be assayed for expression of any or allforms of marker protein (including precursor,endoproteolytically-processed forms, and other forms resulting frompost-translational modification). Nucleic acid may be isolated from adiagnostic sample using standard techniques known to one of skill in theart.

In accordance with the method of the present invention, a diagnosticsample of a subject may be assayed for marker expression, and markerexpression may be detected in a diagnostic sample, using assays anddetection methods readily determined from the known art (e.g.,immunological techniques, hybridization analysis, fluorescence imagingtechniques, and/or radiation detection), as well as any assays anddetection methods disclosed herein (e.g., immunoprecipitation, Westernblot analysis, etc.). For example, a diagnostic sample of a subject maybe assayed for marker expression using an agent reactive with aninflammatory marker. As used herein, “reactive” means the agent hasaffinity for, binds to, or is directed against the marker. As furtherused herein, an “agent” hall include a protein, polypeptide, peptide,nucleic acid (including DNA or RNA), antibody, Fab fragment, F(ab′)₂fragment, molecule, compound, antibiotic, drug, and any combination(s)thereof. Preferably, the agent of the present invention is labeled witha detectable marker or label, in accordance with techniques describedherein. In one embodiment of the present invention, the agent reactivewith a marker is an antibody.

Where the agent of the present invention is an antibody reactive withthe desired marker, a diagnostic sample taken from the subject may bepurified by passage through an affinity column which contains antibodyto the marker, attached as a ligand to a solid support (e.g., aninsoluble organic polymer in the form of a bead, gel, or plate). Theantibody attached to the solid support may be used in the form of acolumn. Examples of suitable solid supports include, without limitation,agarose, cellulose, dextran, polyacrylamide, polystyrene, sepharose, andother insoluble organic polymers. The antibody to the marker may befurther attached to the solid support through a spacer molecule, ifdesired. Appropriate binding conditions (e.g., temperature, pH, and saltconcentration) for ensuring binding of the agent and the antibody may bereadily determined by the skilled artisan. In a preferred embodiment,the antibody to the marker is attached to a sepharose column, such asSepharose 4B.

Additionally, where the agent is an antibody, a diagnostic sample of thesubject may be assayed for expression of the immunological marker usingbinding studies that utilize one or more antibodies immunoreactive withthe marker, along with standard immunological detection techniques. Forexample, the marker protein eluted from the affinity column may besubjected to an ELISA assay, Western blot analysis, flow cytometry, orany other immunostaining method employing an antigen-antibodyinteraction. Preferably, the diagnostic sample is assayed formarkerexpression using Western blotting.

Alternatively, a diagnostic sample of a subject may be assayedfor-marker expression using hybridization analysis of nucleic acidextracted from the diagnostic sample taken from the subject. Accordingto this method of the present invention, the hybridization analysis maybe conducted using Northern blot analysis of mRNA. This method also maybe conducted by performing a Southern blot analysis of DNA using one ormore nucleic acid probes which hybridize to nucleic acid encoding themarker. The nucleic acid probes may be prepared by a variety oftechniques known to those skilled in the art, including, withoutlimitation, the following: restriction enzyme digestion of-markernucleic acid; and automated synthesis of oligonucleotides havingsequences which correspond to selected portions of the nucleotidesequence of the marker nucleic acid, using commercially-availableoligonucleotide synthesizers, such as the Applied Biosystems Model 392DNA/RNA synthesizer.

Nucleic acid probes used in the present invention may be DNA or RNA, andmay vary in length from about 8 nucleotides to the entire length of theinflammatory-marker nucleic acid. In addition, the nucleic acid probesof the present invention may be labeled with one or more detectablemarkers or labels. Labeling of the nucleic acid probes may beaccomplished using one of a number of methods known in the art,including any of those described herein. Combinations of two or morenucleic acid probes (or primers), corresponding to different oroverlapping regions of the marker nucleic acid, also may be used toassay a diagnostic sample for marker expression, using, for example, PCRor RT-PCR.

The detection of marker expression or activity in the method of thepresent invention may be followed by an assay to measure or quantify theextent of marker expression or activity in a diagnostic sample of asubject. Such assays are well known to one of skill in the art, and mayinclude immunohistochemistry/immunocytochemistry, flow cytometry, massspectroscopy, Western blot analysis, or an ELISA for measuring amountsof marker protein or monitoring of substrate production for measuringmarker (enzyme) activity (e.g., DPPIV assay). For example, to use animmunohistochemistry try assay, histological (paraffin-embedded)sections of tissue may be placed on slides, and then incubated with anantibody against a marker. The slides then may be incubated with asecond antibody (against the primary antibody), which is tagged to a dyeor other calorimetric system (e.g., a fluorochrome, a radioactive agent,or an agent having high electron-scanning capacity), to permitvisualization of the marker present in the sections.

The present invention is described in the following Examples, which areset forth to aid in the understanding of the invention, and should notbe construed to limit in any way the scope of the invention as definedin the claims which follow thereafter.

EXAMPLES Example 1 Peptide Synthesis

The peptides in TABLE 1 were synthesized using a solid phase peptidesynthesis technique.

All the required Fmoc-protected amino acids were weighed in three-foldmolar excess relative to the 1 mmole of peptide desired. The amino acidswere then dissolved in Dimethylformaide (DMF) (7.5 ml) to make a 3 mMolsolution. The appropriate amount of Rink amide MBHA resin was weighedtaking into account the resin's substitution. The resin was thentransferred into the automated synthesizer reaction vessel and waspre-soaked with Dichloromethane (DCM) for 15 minutes.

The resin was de-protected by adding 25% piperidine in DMF (30 ml) tothe resin and mixing for 20 minutes. After de-protection of the resinthe first coupling was made by mixing the 3 mMol amino acid solutionwith 4 mMol 2-(1H-benzitriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) and 8 mMol N,N-diisopropylethylamine (DIEPA).The solution was allowed to pre-activate for 5 minutes before beingadded to the resin. The amino acid was allowed to couple for 45 minutes.

After coupling the resin was thoroughly rinsed with DMF andDimethylacetamide (DMA). The attached Fmoc protected amino acid wasdeprotected in the same manner described above and the next amino acidwas attached using the same coupling scheme AA:HBTU:DIEPA.

After the completion of the synthesis the peptide was cleaved from theresin with the use of a cleavage cocktail containing 97.5%Trifluoroacetic acid (TFA) and 2.5% water. The resin was allowed to swimin the cleavage cocktail for 1½ hours. The solution was then filtered bygravity using a Buchner funnel and the filtrate was collected in a 50 mlcentrifugation tube. The peptide was isolated by precipitating withchilled diethyl ether. After centrifuging and decanting diethyl etherthe crude peptide was washed with diethyl ether once more before beingdried in a vacuum desiccator for 2 hours. The peptide was then dissolvedin de-ionized water (10 ml), frozen at −80° C. and lyophilized. The drypeptide was then ready for HPLC purification.

Due to the hydrophilic nature of these peptides the diethyl etherpeptide isolation did not always work. Therefore on occasion achloroform extraction was required. The TFA was evaporated and theresulting peptide residue was dissolved in 10% acetic acid (15 ml). Theimpurities and scavengers were removed from the acetic acid peptidesolution by washing the solution twice with chloroform (30 ml). Theaqueous peptide solution was then frozen at −80° C. and lyophilizedresulting in a powdered peptide ready for HPLC purification.

Peptides SEQ. ID. NOs. 33 and 34 each contained one N-methyl amino acid.This coupling was carried out by combining the N-methyl amino acid,PyBroP and N-hydroxybenzotriazole*H2O(HOBt) and DIEPA solutions togetherin the RV containing the resin. After allowing to couple for 45 minutesthe N-methyl amino acid was then doubled coupled to ensure completecoupling. It was observed that the coupling following the N-methyl aminoacid was not fully complete. Therefore this coupling was performed usingN,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU) instead of HBTU. This still resulted in acrude peptide that typically contained two impurities totaling 30-40% ofthe total purity. The peptide was purified under modified HPLCconditions to isolate the pure peptide peak away from the closelyeluting impurities.

Example 2 Non-Antimicrobial Activity

Bacteria (S. aureus 25923) were seeded into wells containing peptide(200 μM), vehicle (Tris), or antibiotic (erythromycin; 120 μg/ml). Thebacteria were allowed to grow for 2 hours. Thereafter, bacterialviability was determined utilizing a WST-1 colorimetric viability assay(catalogue number 1 644 807; Roche Diagnostics). DMEM and DMEM+WST-1were included as background controls. As shown in FIGS. 1A and B, thepeptide of SEQ ID NOs: 5 and 47 clearly show a lack of activity, ascompared with an antibiotic control.

Example 3 In Vivo Protection

Mice were infected with S. aureus 25923 via intraperitoneal (IP)injection. Four hours later, the peptide of SEQ ID NOs:1, 4, 5, 6, 45,and 47 were administered at 12 mg/kg and 24 mg/kg for SEQ. ID. NO. 1(FIGS. 2A and 2B), 9.6 mg/kg for SEQ. ID. NO. 5 (FIG. 2C), 13 mg/kg forSEQ. ID. NO. 47 (FIG. 2D), 12 mg/kg for SEQ. ID. NO. 4 (FIG. 2E), 9mg/kg for SEQ. ID. NO. 6 (FIG. 2F), and 13 mg/kg for SEQ. ID. NO. 45(FIG. 2G), via IP injection. Twenty-four hours post-infection, survivinganimals were sacrificed, and intraperitoneal lavage fluid was plated todetermine residual bacterial counts (# colony forming units per ml(CFU/ml)) in the presence and absence of peptide treatment.

Dead animals were assigned the highest bacterial count of any animal inthe study. The peptide of SEQ ID NOs:1, 4, 5, 6, 45, and 47 clearlydemonstrated protection, as compared with the control as shown in FIG. 2A-G.

Example 4 Prophylactic In Vivo Protection

Twenty-four hours prior to infection, peptide was administered at 12mg/kg (SEQ. ID. NO. 1, FIG. 3A) and 11.5 mg/kg (SEQ. ID. No. 5, FIG.3B), via IP injection. Mice were then infected with S. aureus 25923 viaIP injection. Twenty-four hours post-infection, surviving animals weresacrificed and intraperitoneal lavage fluid was plated to determineresidual bacterial counts (# colony forming units per ml (CFU/ml)) inthe presence and absence of peptide treatment.

Dead animals were assigned the highest bacterial count of any animal inthe study. The peptides of SEQ. ID. NO:1 and 5 clearly demonstratedprotection ((0 mouse dead (peptide treatment) vs. 2 mice dead(control)). Please see FIGS. 3 A and B.

Discussed below are results obtained by the inventors in connection withthe experiments of Examples 1-4:

The inventors have shown that a peptide having the amino acid sequenceof those shown in TABLE 1 or as described herein as part of theinvention can enhance innate immunity. Specifically, the peptides of SEQID NOs:1, 4, 5, 6, 45, and 47 had the ability to prevent and protectagainst infection, as demonstrated in in vivo models (FIG. 2 and Example3; FIG. 3 and Example 4) However, the peptide of SEQ ID NOs:1, 5 and 47lacked antimicrobial activity, as shown in Example 1 and FIG. 1.Accordingly, modulation of innate immunity, via the peptide of SEQ IDNOs:1, 5 and/or 47, indicate that these peptides can be used as atherapeutic for the treatment of infectious disease.

Example 5 Human Blood Infection Model

Efficacy was also evaluated in an ex vivo model of infection utilizinghuman blood collected by qualified medical personnel from volunteerdonors in heparin tubes. In this model of infection, whole human blood,collected in heparin tubes; was divided into 2 aliquots of 0.5 mL. Eachaliquot had either saline or peptide (0.5 mM) added and was incubatedfor 45 minutes. After incubation, 3.9×10² CFU/mL of S. aureus (ATCCstrain 25923) was added and incubated for 24 hours. After 24 hours,aliquots were taken from each well and used to assess bacterialinfection using enumeration of colony forming units (CFU). The averageresults from multiple CFU enumerations are shown in FIG. 4.

In parallel with efficacy demonstrated in mouse infection models, thishuman ex vivo model further illustrates the efficacy of peptidesidentified in this application for therapeutic applications in humans.

Example 6 Anti-Inflammatory Activity

The effect of peptide treatment on the ex vivo LPS stimulated cytokineresponse was measured. In these studies, SEQ ID NO 5 was administeredintravenously at 100 mg/kg and after 4 hours, blood was collected fromeach mouse. PBMCs were isolated and stimulated with LPS and theresulting cytokine levels were determined. It is apparent from thesestudies that in vivo treatment with peptide is sufficient to change theresponsiveness of the cells to the subsequent, ex vivo stimulation (FIG.5)—resulting in a decreased inflammatory response to LPS stimulation.Similar results are seen with blood collected 24 hours after in vivoadministration.

Example 7 Plasma DPPIV Activity Assay with Mouse Blood

Mouse blood was obtained by cardiac puncture from ICR mice and collectedin heparinized blood collection tubes. Blood from several mice waspooled and aliquoted into 3004 aliquots. The peptide was dissolved inacetate buffered saline, pH 5.5, to a concentration of 9 mM. Of thisstock solution 30 μL were added to 300 μL of blood and mixed byresuspension (concentration in blood 0.82 mM). For the control, 30 μL ofblank acetate buffered saline was added to 300 μL of blood. Each peptidegroup was prepared in triplicate, whereas the control was prepared insix replicates. The samples were incubated at 37° C. in closedmicrotubes for two hours. After incubation the plasma was isolated fromthe samples by centrifugation at 4000 rcf. The plasma was transferred toa 96-well assay plate for the DPPIV assay. The assay was started byadding 5 μL of the DPPIV substrate gly-pro-p-nitroanilide (16 mM inde-ionized water) to 95 μl of plasma (concentration in plasma 0.8 mM)and the increase in UV absorbance (405 nm) was monitored over a 20 mintime period. The rate of the production of p-nitroaniline by enzymaticcleavage of gly-pro-p-nitroanilide was taken as the activity of DPPIV(Durinx C et al., (2001) “Reference values for plasmadipeptidyl-peptidase IV activity and their association with otherlaboratory parameters”. Clin Chem Lab Med. 39(2):155-9.)

The results can be seen in TABLE 1. The effect of the peptides on theactivity of DPPIV was observed. Results are presented as normalized,averaged % activity relative to saline control (set to 100%). Anythingless than 100% activity represents a reduction in DPPIV activity.

In one aspect of the invention, a reduction by of DPPIV activity byabout, or in one embodiment, at least, 25% (i.e. to about 75%+/−5%) wasdeemed to be active. A person skilled in the art would appreciate thatthe desired level of activity may vary depending on the use of thepeptides.

Discussion

The type II transmembrane serine protease dipeptidyl peptidase IV(DPPIV), also known as CD26 or adenosine deaminase binding protein, is amajor regulator of various physiological processes including immunefunctions. CD26/DPPIV is a 110-kD cell surface glycoprotein that ismainly expressed on mature thymocytes, activated T-cells, B-cells,NK-cells, macrophages, and epithelial cells. It has at least twofunctions, a signal transduction function and a proteolytic function(Morimoto C, Schlossman S F. The structure and function of CD26 in .-.The T-cell immune response. Immunol. Review. 1998, 161: 55-70.). One ofits cellular roles involves modulation of chemokine activity by cleavingdipeptides from the chemokine N-terminus. The modulation of the NH₂termini of chemokines is of great importance not only for binding totheir receptors and the following reactions but also for altering thereceptor specificity of the processed chemokine. Furthermore, it wasdemonstrated that soluble rCD26 enhances transendothelial migration of Tcells whereas it reduces the migratory response of monocytes [Oravecz,T. et. al., (1997) Regulation of the receptor specificity and formationof the chemokine RANTES (regulated on activation, normal T cellexpressed and secreted) by dipeptydyl peptidase IV (CD26)-mediatedcleavage. J. Exp. Med. 186:1865-1872; Iwata, S., et. al., (1999)CD26/dipeptidyl peptidase IV differentially regulates the chemotaxis ofT cells and monocytes toward RANTES: possible mechanism for the switchfrom innate to acquired immune response. Int. Int. Immunol. 11:417-426).These results indicate that CD26/DPPIV differentially regulate thechemotactic response of T cells and monocytes and is involved in theswitch from innate to acquired immune response. As such, a reduction inactivity of DPPIV would then have the opposite effect, promoting aninnate immune response and macrophage migratory responses. It has alsobeen reported that pharmacological inhibition of DPPIV enzyme activitycould reduce the progression of arthritis in an experimental rat modelof RA (Tanaka S et al., Anti-arthritic effects of the novel dipeptidylpeptidase IV inhibitors TMC-2A and TSL-225. Immunopharmacology 1998,40:21-26; Tanaka S, et al.: Suppression of arthritis by the inhibitorsof dipeptidyl peptidase IV. Int J Immunopharmacol 1997, 19:15-24),suggesting that decreases in DPPIV-activity may alleviate inflammationunder some circumstances. Together the anti-inflammatory role and itsmodulation of chemokine activity make DPPIV a good molecule forscreening novel compounds for these activities.

CD26/DPPIV is involved in the pathology of a variety of diseases, suchas AIDS and HIV disease progression (Blazquez et al. 1992; Vanham et al.1993; Schols et al. 1998 Oravecz et al. 1995), Graves' disease (Eguchiet al. 1989; Nishikawa et al. 1995), and cancer (Stecca et al. 1997) anddiabetes (Hinke et al. 2000; Marguet et al. 2000).

Further, CD26 as an indicator of T-cell activation has been shown tofluctuate in parallel with several autoimmune diseases such asrheumatoid arthritis (Nakao et al., 1989) and autoimmune thyroiditis(Eguchi et al., 1989). CD26 has been described as a marker thatcorrelates well with the level of activity of these diseases. It hasfurthermore been studied as an indicator of disease progression inchronic progressive multiple sclerosis (Constantinescu et al., 1995).

The peptides of the present invention have demonstrated that they canreduce the activity of DPPIV. As such, they can be used in the treatmentof certain immunological conditions, such as DPPIV-related or associatedconditions, and may, in one aspect modulate innate immunity andinflammation, such as inflammation leading to sepsis.

Example 8 Plasma DPPIV Activity Assay with Human Blood

Human blood was obtained by qualified medical personnel from volunteerdonors and collected in heparinized blood collection tubes. Blood wasaliquoted into 300 μL aliquots. The peptides were dissolved in acetatebuffered saline, pH 5.5. 30 μL of various concentrations were added to300 μL of blood and mixed by resuspension (final concentration in bloodas indicated in FIG. 6). For the control, 30 μL of blank acetatebuffered saline was added to 300 μL of blood. Each concentration wasprepared in triplicate, whereas the control was prepared in sixreplicates. The samples were incubated at 37° C. in closed microtubesfor two hours. After incubation the plasma was isolated from the samplesby centrifugation at 4000 rcf. The plasma was transferred to a 96-wellassay plate for the DPPIV assay. The assay was started by adding 5 μL ofthe DPPIV substrate gly-pro-p-nitroanilide (16 mM in de-ionized water)to 95 μL of plasma (concentration in plasma 0.8 mM) and the increase inUV absorbance (405 nm) was monitored over a 20 min time period. The rateof the production of p-nitroaniline by enzymatic cleavage ofgly-pro-p-nitroanilide was taken as the activity of DPPIV (Durinx C etal., (2001) “Reference values for plasma dipeptidyl-peptidase IVactivity and their association with other laboratory parameters”. ClinChem Lab Med. 39(2):155-9.)

The activity demonstrated with SEQ ID NO. 5 in human blood is comparableto that determined in mouse blood, indicating that the assay results arerelevant for the determination of compounds with therapeutic potentialin humans.

Example 9 Comparative DPPIV Dose Response Curves

Dose response of SEQ. ID. NO. 5 of the present invention and SEQ. ID NO.7 in PCT/CA02/01830, filed Dec. 2, 2002 (KSRIVPAIPVSLL). Peptides wereincubated at different concentrations with whole ICR mouse blood for 2 hat 37° C. Blood incubated with acetate buffered saline (20 mM) was usedas a control. Following incubation, plasma was isolated bycentrifugation for 10 minutes at 4000 rcf. The substrateGly-Pro-p-nitroanilide (0.8 mM) was added to the plasma and theenzymatic rate of DPPIV was determined by monitoring the increase in UVabsorbance of the product p-nitroaniline. The results as illustrated inFIG. 7, indicated a dose response for SEQ. ID. NO. 5 of the presentinvention showing a larger decrease in DPPIV activity with increasingpeptide concentration. A similar dose response was absent forKSRIVPAIPVSLL illustrating that the latter peptide acts distinctly andthat the peptides of the present invention present a novel class ofpeptides.

Example 10 Enhancing the Efficacy of Antibiotic Treatment

Twenty-four hours prior to infection, peptide was administered at 60mg/kg via IP injection to CD-1 mice (N=10 animals/group; FIG. 8A: malemice, FIG. 8B: female mice). Mice were then infected withmethicillin-resistant Staphlococcus aureus (MRSA strain ATCC 33591 inFIG. 8A and UC6685 in FIG. 8B) via IP injection (1.5*10⁷ in FIG. 8A and˜4*10⁵ in FIG. 8B). Vancomycin at the given dosage was administeredtwice subcutaneously 1 and 5 hours post-infection. Survival wasmonitored over 5 (FIG. 8A) or 8 (FIG. 8B) days.

As demonstrated in FIGS. 8 A and B, SEQ ID NO 1 and SEQ ED NO 5 incombination with antibiotic treatment enhanced survival relative to notherapy (vehicle) or antibiotic treatment alone.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art, from a reading of the disclosure, that variouschanges in form and detail can be made without departing from the truescope of the invention in the appended claims.

TABLE 1 all C-terminal amidated unless otherwise indicated**** % DPPIVSEQ Net Activity ID Notes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 * Lengthcharge (Saline)** STDEV 1 + K S R I V P 6 3 76 9 2 Ac denotes Ac K S R IV P 6 2 93 4 acetylation 3 + S R I V P A 6 2 60 4 4 + S R I V P 5 2 645 + R I V P A 5 2 66 6 6 + K I V P A 5 2 64 6 7 *denotes D- + R I V P A*5 2 64 1 amino acid 8 + R V P A 4 2 64 14 9 + R I P A 4 2 51 2 10 Freeacid + R I V P A OH 5 1 51 13 11 + R A V P A 5 2 39 23 12 + R R I V P A6 3 61 3 13 + R K V P A 5 3 50 3 14 + R I V P K 5 3 67 2 15 + R P V P A5 2 27 5 16 + R I P P A 5 2 66 5 17 + R I V P P 5 2 82 4 18 + R I V P GG A 7 2 65 9 19 + G G I V P A 6 1 70 5 20 + G I V P A 5 1 73 8 21 + R GV P A 5 2 64 3 22 + R I V P G 5 2 61 6 23 + R I V P S 5 2 62 10 24 + R IV P L 5 2 74 7 25 + R H V P A 5 2? 38 9 26 + R I P V A 5 2 −4 4 27 + R VI P A 5 2 67 5 28 + R I I P A 5 2 20 3 29 + A V P I R 5 2  0 3 30 + A PV I R 5 2 36 9 31 cyclic head- −R I V P A− 5 1 to-tall 32 cyclic-cysline−C R I V P A C− 7 1 30 4 link 33 x denotes N- + R Ix V P A 5 2 64 10methyl in backbone 34 x denotes N- + R I V P Ax 5 2 72 5 methyl inbackbone 35 + R I V P F 5 2 77 4 36 + Cit I V P A 5 1 81 3 37 + R L V PA 5 2 39 17 38 + H I V P A 5 1? 47 19 39 + I R R V P A 6 3 49 8 40 + A RV P A 5 2 68 7 41 + I R V P A 5 2 49 7 42 + O I V P A 5 2 68 21 43 + S IV P A 5 1 80 8 44 + V S I I K P A R V P S L L 13 3 74 3 45 + K P A R V PS 7 3 27 4 46 + R V P S L L 6 2 70 10 47 + K P R A V P 6 3 52 16 48 + PA R V P 5 2 60 11 49 + I R V P 4 2 56 1 50 + R V P S 8 2 59 1 51 + R V P3 2 70 8 52 + P S V P G S 6 1 74 3 53 + G L K H P S 6 2? 68 6 54 + R I VP A I P V S L L 11 2  4 55 See Note 1 X₁ X₂ P 3 56 See Note 2 X₁ X₂ X₃ P4 57 See Note 3 a X₁ X₂ X₃ P 5 58 See Note 4 X₁ X₂ X₃ P b 5 59 See Note5 a₁ a₂ X₁ X₂ X₃ P 6 60 See Note 6 a X₁ X₂ X₃ P b 6 61 + R I V P A C 6 252 6 62 + r r V P 4 3 51 3 63 hydroxamic + R I V P A NHOH 5 2 acid 64 +R I V P P A 6 2 74 4 65 + R I G P A 5 2 77 4 66 + R I V Pip A 5 2  4 567 + R I V Thz A 5 2 68 68 + R I V Fpro A 5 2 14 69 + R I V Dhp A 5 2 7570 + R I H P A 5 2 37 7 71 + R I W P A 5 2 78 2 72 + R I V P W 5 2 7173 + S P V I R H 6 2 66 8 74 + C P V I R H 6 2 45 3 75 R I E P A 5 1 7910 76 + R I V P E 5 1 69 8 77 + R I V P H 5 1 53 78 + R S V P A 5 2 75 179 + E R I V P A G 7 1 67 9 80 + K V I P S 5 2 30 81 + K V V P S 5 2 575 82 + K P R P 4 3 52 6 83 + R I P 3 2 47 3 84 + O V P 3 2 79 7 85 + S VP 3 1 71 11 86 + K V P 3 2 49 9 87 + R R P 3 3 72 4 88 + G V P 3 1 69 689 + K H P 3 2 72 9 90 *denotes D- R I V P A Y* 6 2 59 5 amino acid **%DPPIV Activity (Saline), where control is 100% activity (saline orvehicle alone without the peptide). ****OH indicates the free acid formof the peptide. Ac indicates acetylated. O indicated Omithine, Citindicated Citrulline, x indicates NMe backbone (versus amide backbone).Note 1 of Table 1: X₁ is selected from the group consisting of K, R, S,O, or glycine based compounds with basic functional groups substitutedon the N-terminal (e.g., Nlys), hSer, Val(betaOH) X₂ is selected fromthe group consisting of V, I, R, and W including an isolated peptide ofup to 10 amino acids comprising an amino acid sequence of SEQ. ID. NO.55. Note 2 of Table 1: wherein X₁ is selected from the group consistingof K, H, R, S, T, O, or glycine based compounds with basic functionalgroups substituted on the N-terminal (e.g., Nlys), hSer, Val(betaOH) andwherein X₂ is selected from the group consisting of A, I, L, V, K, P, G,H, R, S, O, Dab, Dpr, Cit, Hci, Abu, Nva, Nle and where X₂ can beN-methylated, and wherein X₃ is selected from the group consisting of I,V, P, G, H, W, E wherein in one embodiment, X₃ is not N-methylated. Inone embodiment, the isolated peptide can be an amino acid sequence of upto 10 amino acids, but is not SEQ. ID. NOs. 2 or 17. Note 3 of Table 1:wherein X₁, X₂ and X₃ are defined as in Seq. ID. NO. 56, and wherein “a”is selected from the group consisting of S, P, I, R, C, T, L, V, A, G,K, H, R, O, C, M, and F or an isolated peptide up to 10 amino acidscomprising said sequences. Note 4 of Table 1: wherein X₁X₂X₃P are asdefined in SEQ. ID. NO. 56 and “b” is selected from the group consistingof A, A*, E, G, S, L, F, K, W, C, I, V, T, D, Y, R, H, O, Q, N, P and M,but in one embodiment not P. In one embodiment, the isolated peptide isa peptide of up to 10 amino acids comprising SEQ. ID. NO. 58 but notSEQ. ID. NO. 17. Note 5 of Table 1: where X₁, X₂ and X₃ are as definedin SEQ. ID. NO. 56 and “a₁” is selected from the group consisting of K,I R, H, O, L, V, A, and G and “a₂” is selected from the group consistingof S, P, R T, H, K, O, L, V, A, G, S, and I. In one embodiment, “a₁” isnot acetylated, or where a₁ is K, K is not acetylated or not SEQ. ID.NO. 2. In one embodiment, the isolated peptide comprises up to 10 aminoacids comprising SEQ. ID. NO. 59. Note 6 of Table 1: where X₁, X₂ and X₃are as defined in SEQ. ID. NO. 56 and where “a” is selected from thegroup consisting of S, R, K, H, O, T, I, L, V, A, and G and wherein “b”is selected from the group consisting of A, V, I, L, G, K, H, R, O, S,T, and F or a peptide of up to 10 amino acids comprising SEQ. ID. NO.60.

REFERENCES CITED

-   Blazquez M V, Madueno J A, Gonzalez R. Jurado R, Bachovchin W W,    Pena J, Munoz E. Selective decrease of CD26 expression in T cells    from HIV-1-infected individuals. J. Immunol. 1992 Nov. 1;    149(9):3073-7.-   Vanham G, Kestens L, De Meester I, Vingerhoets J, Penne G, Vanhoof    G, Scharpe S, Heyligen H, Bosmans E, Ceuppens J L, et al. Decreased    expression of the memory marker CD26 on both CD4+ and CD8+ T    lymphocytes of HIV-infected subjects. J Acquir Immune Defic Syndr.    1993 July; 6(7):749-57.-   Schols D, Proost P, Struyf S, Wuyts A, De Meester L Scharpe S, Van    Damme J, De Clercq E. CD26-processed RANTES(3-68), but not intact    RANTES, has potent anti-HIV-1 activity. Antiviral Res. 1998 October;    39(3):175-87. Erratum in: Antiviral Res 1999 January; 40(3):189-90.-   Oravecz T, Roderiquez G, Koffi J, Wang J, Ditto M, Bou-Habib D C,    Lusso P, Norcross M A. CD26 expression correlates with entry,    replication and cytopathicity of monocytotropic HIV-1 strains in a    T-cell line. Nat. Med. 1995 September; 1 (9):919-26. Comment in:    Nat. Med. 1995 September; 1(9):881-2.-   Nishikawa Y, Nakamura M, Fukumoto K, Matsumoto M, Matsuda T, Tanaka    Y, Yoshihara H. [Adenosine deaminase isoenzymes in patients with    Graves' disease] Rinsho Byori 1995 October; 43(10):1057-60. [Article    in Japanese]-   Eguchi K, Ueki Y, Shimomura C, Otsubo T, Nakao H, Migita K, Kawakami    A, Matsunaga M, Tezuka H, Ishikawa N, et al. Increment in the Ta1+    cells in the peripheral blood and thyroid tissue of patients with    Graves' disease. J Immunol. 1989 Jun. 15; 142(12):4233-40.-   Stecca B A, Nardo B, Chieco P, Mazziotti A, Bolondi L, Cavallari A.    Aberrant dipeptidyl peptidase IV (DPP IV/CD26) expression in human    hepatocellular carcinoma. J Hepatol. 1997 August; 27(2):337-45.-   Hinke S A, Fospisilik I A, Demuth H U, Mannhart S, Kuhn-Wache I C,    Hoffmann T, Nishimura E, Pederson R A, McIntosh C H. Dipeptidyl    peptidase IV (DPIV/CD26) degradation of glucagon. Characterization    of glucagon degradation products and DPIV-resistant analogs. J Biol.    Chem. 2000 Feb. 11; 275(6):3827-34.-   Marguet D, Baggio L, Kobayashi T, Bernard A M, Pierres M, Nielsen P    F, Ribel U, Watanabe T, Drucker D I, Wagtmann N. Enhanced insulin    secretion and improved glucose tolerance in mice lacking CD26. Proc    Natl Acad Sci USA. 2000 Jun. 6; 97(14:6874-9.-   Nakao H, Eguchi K, Kawakami A, Migita K, Otsubo T, Ueki Y, Shimomura    C, Tezuka H, Matsunaga M, Maeda K, et al. Increment of Ta1 positive    cells in peripheral blood from patients with rheumatoid arthritis. J    Rheumatol. 1989 July; 16(7):904-10.-   Constantinescu C S, Kamoun M, Dotti M, Farber R E, Galetta S L,    Rostami A. A longitudinal study of the T cell activation marker CD26    in chronic progressive multiple sclerosis. J Neurol Sci. 1995 June;    130(2):178-82.-   Kelsen et al., The chemokine receptor CXCR3 and its splice variant    are expressed in human airway epithelial cells, Am. J. Physiol. Lung    Cell Mol. Physiol., 287:L584, 2004-   Morimoto C, Schlossman S F. The structure and function of CD26 in    the T-cell immune response. Immunol. Review. 1998, 161: 55-70-   Tam P J (1988). Synthetic peptide vaccine design: Synthesis and    properties of a high-density multiple antigenic peptide system. Proc    Natl Acad Sci 85, pp. 5409-5413., Briand J P, Barin C, Van    Regenmortel M H V, Muller S (1992)-   J Immunol Meth 156:2, pp. 255-265-   Bundgaard, H., ed., (1985) Design of Prodrugs, Elsevier Science    Publishers, Amsterdam.-   March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New    York (1985) p. 1157-   Mark et al., Encyclopedia of Chemical Technology, John Wiley & Sons,    New York (1980)-   March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New    York (1985) p. 1152-   Mark et al., Encyclopedia of Chemical Technology, John Wiley & Sons,    New York (1980)-   Spatola, A. F. in Chemistry and Biochemistry of Amino Acids,    Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, New    York, p. 267 (1983)-   Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, PEPTIDE    BACKBONE MODIFICATIONS (general review)-   Morley, Trends Pharm Sci (1980) pp. 463 468 (general review)-   Hudson, D. et al., Int J Pept Prot Res 14:177 185 (1979)-   Spatola et al., Life Sci 38:1243 1249 (1986)-   Hann J. Chem. Soc. Perkin Trans. 1307 314 (1982)-   Almquist et al., J Med Chem 23:1392 1398 (1980)-   Jennings-White et al., Tetrahedron Lett 23:2533 (1982)-   Szelke et al., European Application. EP 45665 CA: 97:39405 (1982)-   Holladay et al., Tetrahedron Lett 24:4401 4404 (1983)-   Hruby Life Sci 31:189 199 (1982)

What is claimed is:
 1. An isolated peptide consisting of the amino acidsequence of RRVP (SEQ. ID. NO: 62), or a pharmaceutical salt, ester oramide thereof.
 2. A pharmaceutical composition comprising the isolatedpeptide of claim 1 and a pharmaceutically-acceptable carrier, diluent,or excipient.
 3. The pharmaceutical composition of claim 2 furthercomprising an antibiotic.
 4. The pharmaceutical composition according toclaim 2, wherein the pharmaceutical carrier, diluent, or excipient isone or more selected from the group consisting of carboxymethylcellulose, crystalline cellulose, glycerin, gum arabic, lactose,magnesium stearate, methyl cellulose, powders, saline, sodium alginate,sucrose, starch, talc, and water.
 5. The pharmaceutical compositionaccording to claim 4, wherein the pharmaceutical carrier is saline.